Gene expression system using alternative splicing in insects

Abstract

A polynucleotide expression system is provided that is capable of alternative splicing of RNA transcripts of a polynucleotide sequence to be expressed in an organism.

Claims

1. A polynucleotide expression system capable of expressing a functional protein in an insect, comprising: a heterologous polynucleotide sequence encoding a functional protein, the coding sequence of which is defined between a start codon and a stop codon; a promoter capable of initiating transcription in the insect operably linked to the heterologous polynucleotide sequence; and a splice control sequence, which, in cooperation with a spliceosome in the insect or its offspring, is capable of sex-specifically mediating in the insect or its offspring (i) a first splicing of an RNA transcript of the polynucleotide sequence to produce a first spliced mRNA product, which does not comprise a continuous open reading frame extending from the start codon to the stop codon, and (ii) an alternative splicing of said RNA transcript to yield an alternatively spliced mRNA product, which comprises a continuous open reading frame extending from the start codon to the stop codon, wherein the splice control sequence comprises a core consensus sequence of WWCRAT, wherein W=A or T and R=A or G, or its RNA equivalent and said protein has a lethal effect.

2. The polynucleotide expression system of claim 1, wherein said coding sequence comprises two or more coding exons for the functional protein.

3. The polynucleotide expression system of claim 1, wherein said lethal effect is conditionally suppressible.

4. The polynucleotide expression system of claim 1, wherein said functional protein is selected from the group consisting of an apoptosis-inducing factor, Hid, Reaper (Rpr), and Nipp1Dm.

5. The polynucleotide expression system of claim 1, wherein the functional protein serves as a positive transcriptional control factor for the promoter, such that the functional protein or its expression is controlled by a positive feedback mechanism.

6. The polynucleotide expression system of claim 5, wherein the system further comprises an enhancer associated with said promoter, wherein the functional protein is capable of enhancing activity of the promoter via the enhancer.

7. The polynucleotide expression system of claim 6, wherein the functional protein is a tTA gene product or an analogue thereof and the enhancer comprises one or more tetO operator units operably linked with the promoter.

8. The polynucleotide expression system of claim 1, wherein said functional protein is a transcriptional transactivator selected from the group consisting of tTAV, tTAV2, and tTAV3.

9. The polynucleotide expression system of claim 1, wherein said promoter is activated by environmental conditions.

10. The polynucleotide expression system of claim 1, further comprising an enhancer.

11. The polynucleotide expression system of claim 1, wherein the splice control sequence is derived from a tra intron.

12. The polynucleotide expression system of claim 11, wherein said splice control sequence is derived from the Medfly transformer gene Cctra.

13. The polynucleotide expression system of claim 1, wherein said system comprises a nucleotide sequence selected from the group consisting of SEQ ID NOs: 46-48, 50-56, 106, 143-145, and 151-162.

14. The polynucleotide expression system of claim 1, wherein said splice control sequence is intronic and comprises, on its 5 end, guanine (G) nucleotide, in RNA.

15. The polynucleotide expression system of claim 1, wherein said splice control sequence is intronic and has UG nucleotides flanking its 5 end and GU nucleotides flanking its 3 end, in RNA.

16. The polynucleotide expression system of claim 1, wherein said sex-specific mediation is controlled by binding of a TRA protein, a TRA/TRA2 protein complex, or homolog or homologues thereof.

17. The polynucleotide expression system of claim 1, wherein the splice control sequence is not present in the alternatively spliced mRNA product.

18. The polynucleotide expression system of claim 1, wherein said insect is from the Order Diptera.

19. The polynucleotide expression system of claim 18, wherein said insect is a tephritid fruit fly selected from the group consisting of: Medfly (Ceratitis capitata), Mexfly (Anastrepha ludens), Oriental fruit fly (Bactrocera dorsalis), Olive fruit fly (Bactrocera oleae), Melon fly (Bactrocera cucurbitae), Natal fruit fly (Ceratitis rosa), Cherry fruit fly (Rhagoletis cerasi), Queensland fruit fly (Bactrocera tyroni), Peach fruit fly (Bactrocera zonata), Caribbean fruit fly (Anastrepha suspensa), and West Indian fruit fly (Anastrepha obliqua).

20. The polynucleotide expression system of claim 18, wherein said insect is a mosquito from the genera Stegomyia, Aedes, Anopheles, or Culex.

21. The polynucleotide expression system of claim 20, wherein said mosquito is selected from Aedes aegypti, Aedes albopictus, Anopheles stephensi, Anopheles albimanus, and Anopheles gambiae.

22. The polynucleotide expression system of claim 1, wherein said insect is selected from the group consisting of: the New World screwworm (Cochliomyia hominivorax), the Old World screwworm (Chrysomya bezziana), Australian sheep blowfly (Lucilia cuprina), codling moth (Cydia pomonella), the silk worm (Bombyx mori), the pink bollworm (Pectinophora gossypiella), the diamondback moth (Plutella xylostella), the Gypsy moth (Lymantria dispar), the Navel Orange Worm (Amyelois transitella), the Peach Twig Borer (Anarsia lineatella), the rice stem borer (Tryporyza incertulas), the noctuid moths, Heliothinae, the Japanese beetle (Papilla japonica), White-fringed beetle (Graphognatus spp.), Boll weevil (Anthonomous grandis), corn root worm (Diabrotica spp.), and Colorado potato beetle (Leptinotarsa decemlineata).

23. The polynucleotide expression system of claim 18, wherein said insect is not a Drosphilid.

24. A method of population control of an insect in a natural environment therefor, comprising: i) breeding a stock of the insect, the insect carrying a gene expression system comprising the system of claim 1, wherein the effect of the protein is dominantly lethal; and ii) distributing the said stock insect into the environment, whereby individual stock insects breed with insects of the opposite sex to produce offspring expressing the functional protein.

25. The method of claim 24, wherein the method achieves population control through early stage lethality in one or more of the offspring, which lethality occurs early in development.

26. The method of claim 25, wherein said early stage lethality is embryonic or before sexual maturity.

27. The method of claim 24, wherein expression of the functional protein under the control of a repressible transactivator protein, wherein the breeding is carried out under permissive conditions in the presence of a substance that is absent from the natural environment and capable of repressing said transactivator, whereby the lethal effect is conditional.

28. A method of biological control, comprising: i) breeding a stock of male and female insects transformed with the system of claim 1 under permissive conditions, allowing the survival of male and female insects from the stock; and ii) releasing insects from the stock into the environment at a locus for biological control, whereby individual insects breed with insects of the opposite sex in the wild-type population to produce offspring expressing the functional protein, thereby achieving biological control.

29. A method of sex separation comprising: i) breeding a stock of male and female insects transformed with the expression system of claim 1 under permissive or restrictive conditions, allowing the survival of males and females; and ii) removing the permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not the other by sex-specific alternative splicing of the lethal gene.

30. A method or biological or population control comprising: i) breeding a stock of male and female insects transformed with the gene expression system of claim 1 under permissive or restrictive conditions, allowing the survival of males and females; ii) removing the permissive or restrictive conditions to induce the lethal effect of the lethal gene in one sex and not the other by sex-specific alternative splicing of the lethal gene to achieve sex separation; iii) sterilising or partially sterilising the separated individuals; and iv) achieving said control through release of the separated sterile or partially sterile individuals in to the natural environment of the insect.

31. The polynucleotide expression system of claim 9, wherein the environmental conditions comprise (i) the presence or absence of tetracycline or (ii) temperature.

32. The polynucleotide expression system of claim 1, wherein the functional protein is capable of positively controlling transcription from the promoter.

33. The polynucleotide expression system of claim 1, wherein the splice control sequence comprises the portion of a tra gene that is spliced out in splicing to generate the tra F1 mRNA product.

34. The polynucleotide expression system of claim 33, wherein the tra gene is a non-Drosophilid tra gene.

35. The polynucleotide expression system of claim 34, wherein the tra gene is the Medfly transformer gene Cctra.

36. The polynucleotide expression system of claim 1, wherein: the system further comprises a binding sequence for a repressible transactivator protein, which transactivator protein is repressible by tetracycline or an analog or derivative thereof, wherein the transactivator protein positively controls expression from the promoter; and the splice control sequence comprises the portion of a tra gene that is spliced out in splicing to generate the tra F1 mRNA product.

37. The polynucleotide of claim 36, wherein the tra gene is the Medfly transformer gene Cctra.

38. The polynucleotide expression system of claim 1, wherein the repressible transactivator protein is the functional protein.

39. The polynucleotide expression system of claim 1, wherein: the functional protein is a tTA gene product or an analogue thereof; the system further comprises a tetO operator unit operably linked with the promoter; and the splice control sequence comprises the portion of a tra gene that is spliced out in splicing to generate the tra F1 mRNA product.

40. The polynucleotide expression system of claim 39, wherein the tra gene is the Medfly transformer gene Cctra.

41. The polynucleotide expression system of claim 1, wherein the system comprises the sequence of SEQ ID NO: 51.

42. The method of claim 28, further comprising, prior to step (ii), imposing or permitting restrictive conditions to cause death of insects of one sex, whereby only insects of the other sex are released in step (ii).

43. The polynucleotide expression system of claim 1, wherein the splice control sequence comprises the nucleotide sequence of SEQ ID NO: 1 (TCWWCRATCAACA, wherein W =A or T and R=A or G) or its RNA equivalent.

Description

DESCRIPTION OF THE SEQUENCES OF THE PRESENT INVENTION

(1) SEQ ID NO. 1 tra consensus sequence

(2) SEQ ID NO. 2 LA3097 5 flanking sequence

(3) SEQ ID NO. 3 LA3097 3 flanking sequence

(4) SEQ ID NO. 4 primer 688ie1-transcr

(5) SEQ ID NO. 5 primer 790Aedsx-m-r2

(6) SEQ ID NO. 6 primer 761Aedsx-fem-r

(7) SEQ ID NO. 7 primer AedsxR1

(8) SEQ ID NO. 8 Pane et al consensus sequence

(9) SEQ ID NO. 9 Scali et al 2005 consensus sequence

(10) SEQ ID NOS. 10-33 and 107-138 consensus sequences of putative Tra/Tra2 binding sites deduced for Drosophila (see Table 2).

(11) SEQ ID NO. 34: Open reading frame of tTAV

(12) SEQ ID NO. 35: Protein sequence of tTAV

(13) SEQ ID NO. 36: Open reading frame of tTAV2

(14) SEQ ID NO. 37: Protein sequence of tTAV2

(15) SEQ ID NO. 38: Open reading frame of tTAV3

(16) SEQ ID NO. 39: Protein sequence of tTAV3

(17) SEQ ID NO. 40: Pink Bollworm dsx female specific sequence fragment 1

(18) SEQ ID NO. 41: Pink Bollworm (PBW, Pectinophora gossypiella) dsx female specific sequence fragment 2

(19) SEQ ID NO. 42: Pink Bollworm (PBW, Pectinophora gossypiella) dsx male specific sequence

(20) SEQ ID NO. 43: Partial gene sequence of Aedes aegypti dsx. All exonic sequence is included, but only partial intronic sequencesee FIGS. 47 and 48 for annotation.

(21) SEQ ID NO. 44: Codling moth (Cydia pomonella) dsx female gene sequence: includes a stretch of unknown nucleotides, preferably than then 100, preferably less than 50, more preferably less than 20, more preferably less than 10, and most preferably less than 5.

(22) SEQ ID NO. 45: Codling moth (Cydia pomonella) dsx-male sequence.

(23) SEQ ID NO. 46: Sequence of pLA3435-Bombyx mori-dsx construct/plasmid.

(24) SEQ ID NO. 47: Sequence of pLA3359-Anopheles gambiae dsx construct.

(25) SEQ ID NO. 48: Sequence of pLA3433-Agdsx (Anopheles gambiae) construct with exon 2 included.

(26) SEQ ID NO. 49: Sequence of pLA1188-cctra intron construct

(27) SEQ ID NO. 50: Sequence of pLA3077-a Cctra intron-tTAV construct.

(28) SEQ ID NO. 51: Sequence of pLA3097-a Cctra intron-tTAV construct.

(29) SEQ ID NO. 52: Sequence of pLA3233-Cctra-intron-tTAV2 construct.

(30) SEQ ID NO 53: Sequence of pLA3014-Cctra-intron-Ubiquitin-reaperKR construct.

(31) SEQ ID NO. 54: Sequence of pLA3166-Cctra intron-Ubiquitin-reaperKR construct.

(32) SEQ ID NO. 55: Sequence of pLA3376-Bztra intron-reaperKR and Bztra-intron-tTAV3.

(33) SEQ ID NO. 56: Sequence of pLA3242-Crtra intron-reaperKR construct.

(34) SEQ ID NO. 57: Partial sequence of a male transcript generated in Drosophila melanogaster from LA3077 transformants that differs to the sequence generated in Medfly LA3077 lines. This sequence corresponds to the M3 transcript depicted in FIG. 36.

(35) SEQ ID NO. 58: Partial sequence of Bactrocera zonata tra homologue. Sequence of intron predicted to be spliced out in a female-specific transcript of B. zonata tra (+3 to +970 bp in sequence). Exonic flanking nucleotides are at positions 1-2 and 971-972, i.e. at the 5 and 3 ends of the intronic sequence. In fact, it is worth noting that the intronic sequence is flanked on its 5 end by a Guanine nucleotide, which is thought critical for a clean exit of the intron.

(36) SEQ ID NO 59: Partial sequence of Ceratitis rosa tra homologue. Sequence of intron predicted to be spliced out in a female-specific transcript of C. rosa tra (+3 to 1311 bp in sequence). Exonic flanking nucleotides are present at positions 1-2 and 1312-3. Again, it is noteworthy that the intronic sequence is flanked on its 5 end by a Guanine nucleotide, which is thought critical for a clean exit of the intron.

(37) SEQ ID NOS. 60-70: Primers as referred to in FIGS. 44-46 and 50-51.

(38) SEQ ID NO. 71: Pink Bollworm (PBW, Pectinophora gossypiella) dsx female specific fragment 3.

(39) SEQ ID NO. 72: Open reading frame of Drosophila melanogaster ubiquitin.

(40) SEQ ID NO. 73: Protein sequence of Drosophila melanogaster Ubiquitin.

(41) SEQ ID NOS. 74-105 are primers as discussed above in the Examples.

(42) SEQ ID NO. 106 is the LA1172 nucleotide sequence, including plasmid backbone.

(43) SEQ ID NOs 107-138 are described above.

(44) SEQ ID NO. 139 HSP primer

(45) SEQ ID NO. 140 VP16 primer

(46) SEQ ID NO. 141 primer Agexon1F

(47) SEQ ID NO. 142 primer TETRR1

(48) SEQ ID NO. 143 LA3576 plasmid sequence

(49) SEQ ID NO. 144 LA3582 plasmid sequence

(50) SEQ ID NO. 145 LA3596 plasmid sequence

(51) SEQ ID NO. 146 PBW-dsx (FIG. 6A and FIG. 6B)

(52) SEQ ID NO. 147 bombyx-dsx (FIG. 6A and FIG. 6B)

(53) SEQ ID NO. 148 codling-dsx (FIG. 6A and FIG. 6B)

(54) SEQ ID NO. 149 DSX Minigene1 from construct LA3491

(55) SEQ ID NO. 150 DSX Minigene2 from construct LA3534

(56) SEQ ID NO. 151 LA3619 whole plasmid sequence

(57) SEQ ID NO. 152 LA3612 whole plasmid sequence

(58) SEQ ID NO. 153 LA3491 plasmid sequence

(59) SEQ ID NO. 154 LA3515 plasmid sequence

(60) SEQ ID NO. 155 LA3545 plasmid sequence

(61) SEQ ID NO. 156 LA3604 plasmid sequence

(62) SEQ ID NO. 157 LA3646 plasmid sequence

(63) SEQ ID NO. 158 LA3054 plasmid sequence

(64) SEQ ID NO. 159 LA3056 plasmid sequence

(65) SEQ ID NO. 160 LA3488 plasmid sequence

(66) SEQ ID NO. 161 LA3641 plasmid sequence

(67) SEQ ID NO. 162 LA3570 plasmid sequence

(68) The invention will now be described by reference to the following, non-limiting Examples.

EXAMPLES

Transformer

Example 1Ceratitis capitata Tra Intron

(69) We have prepared an insertion of a Cctra intron cassette into a synthetic open reading frame (ORF). Two versions of this splice correctly in Medfly, in other words the splicing of the Cctra intron cassette faithfully recapitulates what it would normally do in the context of the endogenous Cctra gene. This is to produce 3 (major or only) splice variants in females, one of which is female-specific (called F1), while the other two are found in both males and females (called M1 and M2). Since each of the non-sex-specific transcripts contains additional exonic material with stop codons, we have also arranged this so that only the female splice variant produces functional protein.

(70) Each of these constructs (LA3077 and LA3097) has the Cctra intron flanked by TG and GT (to give 5 . . . TG?intron?GT . . . 3. An older construct, which does not work perfectly, is LA1188. LA1188 is quite well characterizedsplicing is exactly as above except that an additional 4 nucleotides are removed. The intron is in the context 5 . . . TGGCAC?intron?GT . . . 3; splicing removes an additional 4 bases, i.e. 5 . . . TG?GCACintron?GT . . . 3 (FIG. 33).

(71) In all cases the intron is invariant, and is simply the complete Cctra intron sequence. As is normal for introns, it begins GT and ends AG. Almost all introns start with GT, so the use of the rare alternative GC in LA1188 is surprising [GC-AG introns are a known alternativein one large-scale survey, 0.5% of all introns were reported to use GC-AG (Burset et al., 2001), though this may be an underestimate, particularly for alternatively spliced introns, of which perhaps 5% might use GC-AG (Thanaraj and Clark, 2001)].

(72) RT-PCR analysis was performed on LA3077, (a positive feedback construct with the CcTRA intron in the tTAV open reading frame). Transformed adult flies of both sexes were reared on diet substantially free of tetracycline (off tetracycline) for 7 days. Flies were then collected for RNA extraction and RT PCR using primers (HSPSEQ ID NO. 104 and VP16 SEQ ID NO. 105) were used to analyse the splicing pattern of the CcTRA intron (FIG. 34). In two female samples we found the correct splice pattern of the Cctra (776 bp, corresponding to precise removal of the Cctra intron) and saw no such band in males.

(73) We found that LA3077 and LA3097 correspondingly gave repressible female-specific lethality. LA3077 was tested phenotypically through crossing flies heterozygous for LA3077 to wild type, on and off tetracycline. Female lethality ranged from 50 to 70%. LA3097 (a modified version of LA3077 whereby the Cctra intron immediately follows the start codon in the tTAV ORF), demonstrated a much higher level of female specific lethality, peaking at 100% (FIG. 35). The Cctra intron was also inserted in tTAV2 at the same position as LA3097, in construct LA3233, and this gave a similar phenotypic result as LA3097 (FIG. 35).

(74) We have also prepared transformants of LA3077 in Drosophila. Phenotypically, the construct works perfectly, which is to say it is a highly effective female-specific lethal. However, sequencing of the splice variants of one of these insertions has shown that the splicing of this construct in Drosophila is not quite the same as it is in Medfly (SEQ ID NO. 57). The critical transcript, the female-specific one, is the same in both, but at least one of the non-sex-specific transcripts is different. It still incorporates extra exonic sequence, with stop codons, but the splice junctions are not quite the same (FIG. 36). This observation is extremely important in that it shows that this method (regulation of gene expression by use of alternatively spliced introns) can be used across quite a wide phylogenetic range.

(75) A simple test to determine whether an as yet uncharacterized exonic splice regulator (such as enhancers and suppressors) may be modifying the function of the alternatively spliced intron, could include making the construct and introducing it into a target tissue, then examining its splice pattern. In many cases this will not require germline transformation, so the test can be quite rapid, for instance by transient expression in suitable tissue culture cells or in vivo. For instance, in vivo testing in insects could be achieved by delivering the DNA by microinjection. However, as the skilled person will appreciate, microinjection coupled with electroporation, or electroporation, chemical transformation, ballistic methods, for instance, have all been used in a number of various contexts and such methods of plasmid introduction and protein expression therefrom are well known in the art.

(76) We have also recently made, and have obtained transgenics with, the Cctra intron in a different gene (LA3014) (all the above examples are in tTAV). LA3014 contains a ubiquitin-reaper.sup.KR fusion downstream of a Cctra intron. Phenotypic data (FIG. 35) shows that LA3014 transgenic Medfly gave repressible female-specific lethality. RT-PCR analysis on RNA extracted from adult males and females raised off tetracycline, using primers (HSP, SEQ ID NO 74) and ReaperKR (SEQ ID NO. 75), demonstrate that correct splicing was occurring in females (508 bp band) and no such band was found in males (FIG. 37). LA3166 is another construct with the Cctra intron placed inside the ubiquitin coding region fused to reaper.sup.KR, but placed in a different position in ubiquitin. LA3166 also produces a dominant repressible female-specific lethal effect in Medfly (FIG. 35).

(77) We have also recently made, and have obtained transgenics with, intron-only Cctra-based constructs with the intron in a different gene (all the above examples are in tTAV or one of its variants, i.e. tTAV2 or tTAV3). These constructs work as predicted. This is an important result, thus showing that there are not essential exonic sequences in Cctra that we have simply duplicated (in function, if not necessarily in sequence) by chance, in tTAV. We also have ubi-rpr.sup.KR constructs of this type (LA3014 and LA3166), which also validates the ubiquitin fusion method described above.

(78) In order to demonstrate the phylogenetic range of the Cctra intron we generated transgenic LA3097 and LA3233 Anastrepha ludens. LA3097 and LA3233 were selected for injection into Anastrepha ludens as they demonstrated the best female specific lethality in Ceratitis capitata (see Example 13). Phenotypic data was generated for 4 independent LA3097 lines and 1 LA3233 line (see FIG. 38). Female specific lethality was generally somewhat lower in Anastrepha ludens when compared to C. capitata but reached 100% in one line.

(79) Anastrepha ludens transformed with LA3097 and raised on tetracycline until eclosion were isolated and maintained off tetracycline for 7 days. RNA was then extracted and RT-PCR analysis was performed using primers HSP (SEQ ID NO. 76) and TETRR1 (SEQ ID NO. 77). The correct female specific (F1-like) splice pattern was observed RNA isolated from in females (348 bp) but not from males demonstrating the function of the Cctra intron in a different species (FIG. 39)

(80) The brightest male band and the female specific band were purified and precipitated for sequencing. The female specific transcript was found to be correctly spliced in Mexfly females as expected for LA3097:

(81) TABLE-US-00001 LA3097: AGCCACCATG?? GT...intron...AG? GTCAGCCGCC

(82) The two flanking sequences above are SEQ ID NOS. 2 and 3, respectively.

Example 2Bactocera zonata Tra Intron

(83) We isolated the tra intron from Bactocera zonata (B. zonata) (SEQ ID NO. 58) using primers ROSA1 (SEQ ID NO. 78), ROSA2 (SEQ ID NO. 79), and ROSA3 (SEQ ID NO. 80).

(84) These primer sequences were designed based on conserved coding sequence of Ceratitis capitata and Bactrocera oleae tra homologs. Using ROSA2 and ROSA3 or ROSA1 and ROSA3 as primers, the tra intron and its flanking coding region were amplified from Bactrocera zonata genomic DNA. Then we used these PCR products as a template and amplified the tra intron fragment to make the construct-LA3376 (FIG. 31 and SEQ ID NO. 55). The primers (BZNHESEQ ID NO. 81 and BZR-SEQ ID NO. 82) were used for making the constructs; these primers contain additional sequences for cloning purposes. The Bztra intron in LA3376 is cloned into the ORF of tTAV3 and also of reaper.sup.KR. Medfly transformants were generated and RNA extracted from male and female flies.

(85) RT-PCR was then performed on both the reaper.sup.KR (HBSEQ ID NO. 83) and Reaper KRSEQ ID NO. 84) and tTAV3 (SRYSEQ ID NO. 85) and AV3FSEQ ID NO. 86) splice. The expected fragments of 200 bp for reaper.sup.KR and 670 bp for tTAV3, corresponding to splicing in a pattern equivalent to the F1 transcript of Cctra (Pane et al., 2002), were generated in females (FIG. 40).

Example 3Isolation and Splicing of the Ceratitis rosa (C. rosa, Natal Fruit Fly) Tra Intron

(86) Primers ROSA2 (SEQ ID NO. 87) and ROSA3 (SEQ ID NO. 88) were designed based on conserved coding sequence of Ceratitis capitata and Bactrocera oleae Using ROSA2 and ROSA3 as primers, the tra intron and its flanking coding region were amplified from Ceratitis rosa genomic DNA (SEQ ID NO. 59). We then used the PCR products as a template and amplified the tra intron fragment to make constructs. The primers (CRNHESEQ ID NO 89 and CRR SEQ ID NO 90) were used during the construction of LA3242 (SEQ ID NO. 56 and FIG. 32. LA3242 contains the C. rosa intron at the 5 end of the reaper.sup.KR ORF. Ceratitis capitata embryos were injected with DNA of LA3242, injected embryos were raised to adulthood on a diet substantially free of tetracycline. RNA was extracted from adult males and females; this was used as a template for RT PCR using primers HB (SEQ ID NO. 91) and ReaperKR (SEQ ID NO. 92). The expected female-specific splice band (200 bp), corresponding to splicing in the equivalent pattern to that of transcript F1 of Cctra, was observed in females and not males (FIG. 41).

Double-Sex

Example 4Bombyx mori Dsx in PBW

(87) The sequence of a Bombyx mori (silk moth) homolog of Drosophila Dsx (Bmdsx) has been previously described and a male- and a female-specific splice product have been identified (Suzuki et al, 2001). Both males and females use the same 3 polyA, and there are two female specific exons. One paper has suggested that the sex-specific splicing is not dependent on tra/tra2, in other words even though the pattern looks the same, the underlying mechanism may be different (Suzuki et al., 2001), though their data, principally the lack of recognisable tra-tra2 binding sites, however, is not compelling. In addition, a B. mori dsx mini-gene construct (containing exonic sequence and truncated intronic sequence) has been transformed into B. mori and the germline transformants show sex-specific splicing (Funaguma et al., 2005).

(88) We have generated a Bmdsx minigene based on the sequence used in the Funaguma et al paper, with some significant changes, and injected this into the moth Pink Bollworm to ascertain if one can obtain sex-specific splicing in a divergent species. The mini-gene construct we generated does not included exon 1, which is present in both males and females. In addition, we removed the intron between exon 3 and 4 (the two female specific exons), included a heterologous sequence (containing multiple cloning sites, MCS), used the Hr5-IE1 enhancer/promoter sequence from the baculovirus AcNPV and used a 3 transcriptional termination sequence derived from SV40 (see FIG. 42 for a schematic). The individual exon/flanking intron fragments used were amplified and recombined together by PCR and ligated into a construct carrying a Hr5/IE1 enhancer promoter fragment and SV40 3UTR (FIG. 22 and SEQ ID NO. 22).

(89) LA3435 was injected into pink bollworm (Pectinophora gossypiella) embryos. First instar larvae were collected after 5-7 days and analysed individually by RT-PCR (using primers IE1 transcrSEQ ID NO. 93 and SV40-RT-P2SEQ ID NO. 94) to determine if BMdsx can undergo male and female specific splicing (FIG. 43). Our analysis detected the male specific band (predicted to be 442 bp) in 4 samples (Lanes 1, 2, 3 and 4) and the female specific band (predicted to be 612 bp) in 1 sample (Lane 5).

(90) The correct splicing of B. mori dsx in PBW demonstrates that we can achieve (have achieved) sex-specific expression of a heterologous sequence (here, the MCS) in a Lepidopteran by utilizing an alternative splicing system. Furthermore, since this splicing system was derived from a heterologous species, this suggests that such constructs might work over a wide phylogenetic range. However, the identification of alternative splicing systems in the species of interest is also envisioned, and methods for identifying such alternative splicing systems are provided herein or will be known to the person skilled in the art. By providing a MCS in our Example (see FIG. 42), the expression of a sequence of interest, for example a coding region for a protein of interest could readily be achieved by inserting said sequence. If said sequence encoded a suitable protein, a sex-specific phenotype, for example conditional sex-specific lethality, could thereby be introduced, for example into pink bollworm.

Example 5Isolation of Codling Moth Dsx

(91) The dsx gene from Codling moth (Cydia pomonella) was isolated by performing 3 RACE using primers which were based on sequence alignments from B. oleae, B. tyroni, C. capitata, D. melanogaster, B. mori, and A. gambiae. RNA was isolated from a male and female codling moth and 3 RACE, to generate cDNA, was performed using the TT7T25 primer (SEQ ID NO. 95).

(92) PCR was performed using the primers ds1c (SEQ ID NO. 96) and TT7 (SEQ ID NO. 97). Two rounds of nested PCR were then performed on the product of the first PCR using the primers codling2a (SEQ ID NO. 98) and TT7 (SEQ ID NO. 99) and the product of the second round of PCR using Codling2b (SEQ ID NO. 100) and TT7. The isolated male and female specific sequences share sequence similarity to previously isolated dsx homologues (Male-SEQ ID NO. 43 and FemaleSEQ ID NO. 42).

Example 6Isolation of PBW Dsx

(93) The dsx gene from pink bollworm was isolated by performing 3 RACE using primers which were based on sequence alignments from B. oleae, B. tyroni, C. capitata, D. melanogaster, B. mori, and A. gambiae. RNA was isolated from a male and female codling moth and 3 RACE, to generate cDNA, was performed using TT7T25 (sequence defined herein). PCR was performed using the primers Pbwdsx2 (SEQ ID NO. 101) and TT7 (SEQ ID NO. 102). Nested PCR was then performed on the product of the first PCR using the primers Pbwdsx3 (SEQ ID NO. 103) and TT7. Three female specific sequences were isolated: PBWdsx-F1 (SEQ ID NO. 40), PBWdsx-F2 (FIG. 10), and PBWdsx-F3 (SEQ ID NO. 71) and one male specific sequence (SEQ ID NO. 42). The isolated male and female specific sequences share sequence similarity to previously isolated dsx homologues.

Example 7Dsx in Anopheles gambiae

(94) The sequence of the dsx gene of Anopheles gambiae has previously been described (Scali et al 2005). However, when we have tried to repeat the work described in the paper we find that there are some differences in the splicing that occurs. When we tried to repeat the amplification of the female specific transcript using primers designed from the mRNA sequence (Accession; AY903308 for female coding sequence and AY903307 for male coding sequence), the amplification failed. However, when Scali and colleagues showed that there was a shared exon, which had previously not been described, we designed primers to amplify the entire dsx transcript and gene. Using these primers and primers designed from genomic DNA sequence (Accession; GI:19611767) we find that the splicing of the female transcript is different from that described by Scali et al 2005 (FIG. 44). The transcript showed that the female exon was in a different position. There are several explanations for these differences, but the most likely are either some sort of strain difference in the Anopheles that we used to get the data from, or the published sequence is not from Anopheles gambiae, or there is more than one female isoform as shown for Stegomyia aegypti in Example 20.

(95) We have also successfully used primers, designed around our version of the Anopheles gambiae dsx splicing, that are able to distinguish between males and females of Anopheles gambiae (FIG. 45). This provides good evidence that the system will be functional as a sex-specific splicing mechanism when fused to a protein of interest, such as tTAV or a killer.

(96) The Anopheles gambiae dsx gene that we have isolated from genomic DNA, which has several changes in nucleotide sequence compared to the reported genomic sequence, was cloned into LA3359 (SEQ ID NO. 47) and LA3433 (SEQ ID NO. 48), schematics can be found in FIG. 23 and FIG. 24, respectively.

Example 8Dsx in Stegomyia aegypti

(97) The splicing of the gene appears to be similar to Anopheles gambiae dsx (Scali et al 2005). The Stegomyia aegypti dsx gene is illustrated diagrammatically in FIG. 47 or 48. A male-specific transcript (M1) is produced which does not include exons 5a or 5b. Two female specific splice variants (F1 and F2) have the following structure; F1 comprises exons 1-4, 5a, 6 and 7 but not 5b, F2 comprises exons 1-4 and 5b (FIG. 46). In addition, a further transcript (C1) is present in both males and females; this comprises exons 1-4 and 7, but not exons 5a, 5b or 6.

(98) The splicing of the gene appears to be similar to Anopheles gambiae dsx (Scali et al 2005). The Stegomyia aegypti dsx gene is illustrated diagrammatically in FIG. 47 or 48.

Actin 4

Example 9Stegomyia aegypti Actin-4 Gene

(99) One way to get sex-, tissue- and stage-specific expression of a gene of interest is to link it with the Stegomyia aegypti Actin-4 (AeAct-4) gene. This gene is only expressed in the developing flight muscles of female Stegomyia aegypti (Munoz et al 2004). They used in-situ hybridisation to an RNA to detect the expression profile of AeAct-4. We have taken a fragment of the Stegomyia aegypti Actin-4 gene, comprising a putative promoter region, an alternatively spliced intron, and a section of 5 untranslated region (UTR) and placed it in front of sequence coding for tTAV (FIG. 49) to test the function of the sex specific splicing when fused to tTAV.

(100) We integrated LA1172 into the Stegomyia aegypti genome using piggyBac. Two independent lines were generated (lines 2 and 8). Both of these lines show the correct splicing of the Actin-4-tTAV gene (FIGS. 50 and 51). The Actin-4 promoter and alternatively spliced intron can therefore be used successfully to provide sex-, tissue- and stage-specific splicing of a gene of interest in Stegomyia aegypti.

Description of the Figures and Sequence Listings of Examples 1-9

(101) FIG. 19: One use of the P element in generating germline-specific expression of a gene of interest (Gene E).

(102) Insertion of the P element IVS3 and flanking exonic sequences upstream of an ubiquitin-Gene E fusion with allow germline-specific expression of Gene E under a germline active promoter. AGermline active promoter; BP-element open reading frame; CP intron IVS3; DUbiquitin; ECoding region for protein of Interest e.g. tTAV.

(103) FIG. 20: Sex-specific expression using dsx.

(104) A: Intron used as Cctra intron above, but giving male-specific expression. A fragment of dsx (here the Anopheles version) is inserted into a heterologous coding region (shaded boxes). The intron is completely removed in males, but in females the coding region is prematurely terminated.

(105) B: An alternative approach to male-specific expression, in which a heterologous coding region is fused to a fragment of dsx.

(106) C: Female-specific expression: the heterologous coding region is inserted into the female-specific exon, either as an in-frame fusion to a fragment of Dsx, or with its own start and stop codons.

(107) D: Differential expression: designs B and C can be combined to give expression of gene a in females and b in males.

(108) FIG. 21: Sex-specific alternative splicing of Cctra

(109) A: Cctra is spliced in females to produce three transcripts: F1, which encodes functional Tra protein, and M1 and M2, which do not, because they include additional exons with stop codons (redrawn from Pane et al. 2002). Males produce only transcripts M1 and M2 and therefore do not produce functional Tra protein at all.

(110) B: If this intron were to function similarly in a heterologous coding region, this would similarly allow females, but not males, to produce functional protein X.

(111) FIG. 22: Diagrammatic representation of pLA3435 construct/plasmid (SEQ ID NO. 46).

(112) FIG. 23: Plasmid map of pLA3359 Anopheles gambiae dsx gene placed under the control of a Hr5-IE1 promoter for assessing splicing via transient expression.

(113) FIG. 24: pLA3433-Anopheles gambiae dsx gene placed under the contron1 of a Hr5-IE1 promoter, with the addition of exon 2, for assessing splicing via transient expression.

(114) FIG. 25: Schematic representation of pLA1188 construct.

(115) FIG. 26: Schematic diagram of pLA3077 construct.

(116) FIG. 27: Schematic diagram of pLA3097 construct.

(117) FIG. 28: Schematic diagram of pLA3233 construct.

(118) FIG. 29: Schematic diagram of pLA3014 construct.

(119) FIG. 30: Schematic diagram of pLA3166 construct.

(120) FIG. 31: Schematic diagram of pLA3376 construct.

(121) FIG. 32: Schematic diagram of pLA3242 construct.

(122) FIG. 33: Flanking sequence of Cctra

(123) Splicing of the Cctra intron in LA3077 and LA3097 is exactly as you would see in the native Cctra intron. Splicing in LA1188 results in the removal of 4 additional nucleotides. In all cases the introns are flanked by 5 exonic TG and 3 GT. The sequences flanking the GT . . . intron . . . AG in LA3097 are given in SEQ ID NO:2 and SEQ ID NO:3. The sequences flanking the GT . . . intron . . . AG in LA3077 are given in SEQ ID NO:163 and SEQ ID NO:164, the sequences flanking the GT . . . intron . . . AG in LA1188 are given in SEQ ID NO:165 and SEQ ID NO:166, and the sequences flanking the GT . . . intron . . . AG in the native are given in SEQ ID NO: 167 and 168.

(124) FIG. 34: Gel showing correct sex-specific splicing of intron(s) derived from CcTra (776 bp band in females) in Ceratitis capitata transformed with LA3077. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.8, 1.0 and 1.5 kb are indicated); Lanes 2 and 3: Ceratitis capitata LA3077/+ males; Lanes 4 and 5: Ceratitis capitata LA3077/+ females.

(125) FIG. 35: Phenotypic data for transformed female specific constructs in Ceratitis capitata. Column 1: Construct designation LA#, e.g. LA3077, LA3097, LA3233, etc, is indicated by number, with independent insertion lines referred to by letter; Columns 2 and 3: Non-tetracycline (NT) results for each transformed line given in total males (2) and total females (3). Columns 4 and 5: Tetracycline (TET) results for each transformed line given in total males (4) and total females (5).

(126) FIG. 36: Transcripts of Cctra intron constructs in Drosophila and Ceratitis capitata.

(127) The top line represents the construct DNA containing tra intron flanked by desired gene (the open box). The red box represents the male specific exons. Introns are represented by solid lines. Arrow above the first line represents the positions of the oligonucleotides used in the RT-PCR experiments. The bar indicates the scale of the figure.

(128) FIG. 37: Gel showing correct female specific splicing of CcTRA-derived sequence (508 bp band) in female Ceratitis capitata transformed with LA3014. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.4 and 1.0 kb are indicated); Lane 2 Ceratitis capitata LA3014/+ male; Lane 4: Ceratitis capitata LA3014/+ female; Lanes 3 and 5: no reverse transcriptase negative controls (background bands, probably from genomic DNA, can be seen in lanes 2 and 4).

(129) FIG. 38: Phenotypic data for transgenic Anastrepha ludens transformed with LA3097 or LA3233. Column 1: Construct LA# (LA3097 or LA3233) indicated, with independent insertion lines referred to by letter; Columns 2 and 3: Non-tetracycline (NT) results for each transformed line given in total males (2) and total females (3). Columns 4 and 5: Tetracycline (TET) results for each transformed line given in total males (4) and total females (5).

(130) FIG. 39: Gel showing correct sex-specific splicing of CcTRA splicing (348 bp band in females) in Anastrepha ludens transformed with LA3097. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.4 and 1.0 kb are indicated); Lanes 2, 3 and 4: A. ludens LA3097/+ males; Lanes 5, 6 and 7: A. ludens LA3097/+ females.

(131) FIG. 40: Gel showing correct sex-specific splicing of BzTRA in reaperKR (200 bp band in females) and tTAV3 (670 bp band in females) regions of LA3376, in Ceratitis capitata transformed with LA3376. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.2, 0.6 and 1.0 kb are indicated); Lanes 2 and 3: C. capitata LA3376/+ males tested for splicing in reaperKR; Lanes 4 and 5: C. capitata LA3376/+ females tested for splicing in reaperKR; Lane 6: SmartLadder?; Lanes 7 and 8: C. capitata LA3376/+ males tested for splicing in tTAV; Lanes 9 and 10: C. capitata LA3376/+ females tested for splicing in tTAV; Lane 11: SmartLadder?

(132) FIG. 41: Gel showing correct sex-specific CrTRA splicing in CrTRA-reaperKR (200 bp band in females) in Ceratitis capitata injected with LA3242. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.2, 0.6 and 1.0 kb are indicated); Lanes 2-7: C. capitata wild type males injected with LA3242; Lane 8: SmartLadder?; Lanes 9-14: C. capitata wild type females injected with LA3242; Lane 15: SmartLadder?.

(133) FIG. 42: Schematic representation of Bmdsx minigene constructs.

(134) Two minigene constructs derived from the Bombyx mori dsx gene are illustrated diagrammatically, together with the predicted alternative splicing of these constructs (female pattern shown above the construct, male pattern below). (A) is the Bombyx mori dsx mini-gene construct used in Funaguma et al., 2005) (B) is pLA3435. A and B differ from each other in several ways: (i) Exon 1 is excluded from pLA3435, (ii) the intron between female specific exons 3 and 4 has been removed and a short heterologous sequence has been inserted in pLA3435 (iii) Funaguma et al., use the ie1 promoter from the baculovirus BmNPV and a BmA3 3UTR compared with pLA3435 which uses the hr5-IE1 enhancer/promoter from the baculovirus AcNPV and a 3 SV40 3UTR. (iv) pLA3435 uses slightly longer intron sequences when compared with (A) (see FIG. 15 for sequence). Two minigene constructs derived from the Bombyx mori dsx gene are illustrated diagrammatically, together with the predicted alternative splicing of these constructs (female pattern shown above the construct, male pattern below).

(135) FIG. 43: Sex-specific splicing of BMdsx mini-gene construct in PBW.

(136) Analysis of transient expression from pLA3435 using RT-PCR show the presence of a 442 bp fragment (Lanes 1,2,3 and 4) in males and a 612 bp fragment in females (Lane 5), showing that the BMdsx mini-gene with a heterologous fragment inserted between exon 3 and 4 is able to splice correctly in the divergent moth, PBW. Markers are SmartLadder? from Eurogentec; bands of approx 0.2, 0.4 and 0.6 kb are indicated

(137) FIG. 44: Sex-specific splicing of Anopheles gambiae dsx.

(138) Anopheles (A) shows the splicing that was reported by Scali et al 2005. However, when RT-PCR was performed using our primers (spl-agdsx-e3 (SEQ ID NO. 60) and spl-agdsx-m (SEQ ID NO. 61)) a different splicing pattern for females was revealed, represented by Anopheles (B).

(139) FIG. 45: Identification of male and female Anopheles gambiae using dsx primers.

(140) RNA was extracted from male and female Anopheles gambiae and the dsx transcripts were amplified by RT-PCR using the primers spl-agdsx-e3 (SEQ ID NO. 62) and spl-agdsx-m (SEQ ID NO. 63); the resulting banding pattern is shown in the gel above. The expected bands for the male and female transcripts are indicated by the white arrows, the bands have been cloned and sequenced and are identical to the predicted sequence of our version of the dsx transcript (see SEQ ID NO. 47 (LA3359) and SEQ ID NO. 48 (LA3433)). The molecular weight markers are shown in kb (SmartLadder? from Eurogentec; sizes are approximate).

(141) FIG. 46: Identification of male and female Stegomyia aegypti using dsx primers.

(142) The primers for the Stegomyia aegypti RT-PCR for A and B were aedesxF1 (SEQ ID NO. 64) and aedesxR5 (SEQ ID NO. 65) were tested initially on pupae, a life stage of Stegomyia aegypti that can be sexed conveniently and accurately; the resulting RT-PCR amplification is shown on gel image (A). The male and female pupae show a distinctive sex specific band. Then the primers were tested on RNA extractions from larvae, which can not be readily sexed by their morphology and the resulting RT-PCR amplification shown on gel image (B). The larvae show a clear banding pattern which distinguishes males from females unambiguously. Gel image (C) shows an approximately 600 bp band from RT-PCR using the primers aedessxF1 and aedesxR2 (SEQ ID NO. 66) from individual male and female pupa. Sequencing of this band showed a female specific splice variant which does not appear to possess the male shared exon to which aedesxR5 is predicted to anneal (exon 7, see FIG. 56). The molecular weight markers are shown in kb (SmartLadder? from Eurogentec; sizes are approximate).

(143) FIG. 47: Diagrammatic representation of part of the Stegomyia aegypti dsx gene (not to scale).

(144) A fragment of the Stegomyia aegypti dsx gene is represented above. Exons 5a and 5b are female specific and exon 6 is a male specific exon. Two female-specific splice variants have been found (F1 and F2) which comprise exons 1-4,5b,6 and 7 (F1) or 1-4,5a (F2); transcripts in males (M1) comprise exons 1-4,6 and 7 but not exon 5a or 5b and a transcript (C1) of 1-4 and 7 but not exons 5a, 5b or 6 is shown in males and females. The numbers for each of the exons after #relates to contig 1.370, see internet address broad.mit.edu/annotation/disease_vector/aedes_aegypti/, which reads in the opposite orientation, and after * relate to the nucleotide sequence shown in SEQ ID NO. 43.

(145) FIG. 48: Diagrammatic representation of the Stegomyia aegypti dsx gene.

(146) The entire Stegomyia aegypti dsx gene is represented above Exon 5 is the female specific exon and exon 6 is a putative male specific exon. In principle, transcripts in females comprise exons 1, 2, 3, 4, 5, and 7, and males comprise exons 1, 2, 3, 4, 6, and 7. The numbers for each of the exons after #relates to contig 1.370, see internet address broad.mit.edu/annotation/disease_vector/aedes_aegypti/, reading in the opposite orientation, and after * relate to FIG. 12.

(147) FIG. 49: Plasmid map of pLA 1172.

(148) A coding region for tTAV has been placed under the control of a fragment from the Stegomyia aegypti actin-4 gene (Munoz et al 2005) which includes the 5 UTR, first intron, and upstream sequences (putative promoter). The construct also contains a tetO.sub.7 Nipper sequence. The construct has piggyBac ends and a DsRed2 marker for stable integration into a genome.

(149) FIG. 50: Sex-specific splicing of tTAV in LA1172 transformants.

(150) Gel image of RT-PCR of RNA extracted from LA1172 line 2 male and female pupa. The primers used were Agexon1 (SEQ ID NO. 67) and Tra (tTAV) seq+ (SEQ ID NO. 68). Sequencing of the RT-PCR bands showed the expected splicing occurring in males and females. The data shown in the above diagram is for LA1172 line 2, line 8 showed exactly the same results (data not shown). Markers are SmartLadder? from Eurogentec; approximate sizes are indicated, in kb).

(151) FIG. 51: RT-PCR of wild type samples, showing sex-specific splice variants of the Stegomyia aegypti Actin-4 gene.

(152) Gel image of RT-PCR of RNA extracted from different developmental stages, and dissections of adults, of LA1172 line 8. The primers used were Agexon1 (SEQ ID NO. 69) and Exon 3 (SEQ ID NO. 70). The gel image shows that strong expression from the Actin-4 gene only occurs at the pupal stage, and that adult expression is generally limited to the female thorax where the flight muscles are found. Table 17, below show the contents of each lane.

(153) TABLE-US-00002 TABLE 1 E = pool of ~100 embryos L4 = 4.sup.th instar larva ME = early male pupa (<4 hours old) FE = early female pupa (<4 hours old) MP = male pupa FP = female pupae MH = head from male adult MT = thorax from male adult MA = abdomen from male adult FH = head from female adult FT = thorax from female adult FA = abdomen from female adult ?ve = water control

FURTHER EXAMPLES

Example 10: Moths

(154) We have newly made constructs based on our transient expression data using a recombinant minigene construct derived from Bombyx mori. This is discussed further below in the section entitled Moth dsx sequence alignment and conserved motifs

Example 11: Use of Bztra

(155) We have newly made two Bztra-based constructs, expressed in Mexfly (LA3376). LA3376 gives repressible female-specific lethality. LA3376 we have previously shown to function and splice correctly in Medfly. Transformants in Mexfly (Anastrepha ludens) were also generated with LA3376. These were analysed for correct splicing of the Bztra intron in order to demonstrate the phylogenetic range of the Bztra intron by RT-PCR using primers SRY and AV3F (FIG. 15 and Medfly RT-PCR gels section above). This shows correct splicing of the Bztra intron in Mexfly.

Example 12: Dmdsx in Medfly (DmDsx in Transgenic Medfly Example: Nipper Fusion in #797)

(156) We also have newly made data on a Dmdsx construct in Medfly. The construct used a fragment of the Drosophila melanogaster gene doublesex to give sex-specific expression of a fragment of the Drosophila melanogaster gene Nipp1Dm (we call this fragment nipper). We didn't see clear sex-specific splicing. However, the phenotypic data shows some sex-specificity; we saw increased lethality of females, to about 75% penetration. Of course this incomplete penetrance could be due to expression level, lack of toxicity of nipper in Medfly, etc. We also had a significant reduction in the number of males, but the tTA source, LA670, used in this experiment could itself be killing some of the males.

(157) We have tested three independent Medfly transgenic lines that carry a fusion of nipper to DmDsx sequence that was intended to be expressed specifically in females. This construct may not have worked perfectly possibly due to essential sequence for correct alternative splicing and/or the Sxl binding sites required by DmDsx, and since Medfly do not use Sxl in the sex-determining pathway, DmDsx may be unable to completely splice this fusion in the correct way in Medfly. However, we were successful in reproducibly causing increased lethality in females compared to males across all three lines at a very similar efficiency (approximately 75% more lethality observed in females than in males). This demonstrates the dsx system can work across quite distantly related species (evolutionary separation is around 120-150 Million years), and if the Ccdsx sequence were used it may have well worked due to the Sxl requirement of Dmdsx.

(158) The 797 results are shown below, using a Tet014 dsx splice nipper (Pub EGFP) system. They show that this system is lethal at the larval stage (50%), and is likely to be acting more successfully in females (75%). 797 is marked with green (G), 670 with red (R). 670 is a tTAV source, so one expects to see a phenotype in the R+G flies; G (and R) only are controls. NFnon-fluorescent (i.e. wild type) is also a control where included. All progeny reared on tet-free media.

(159) All three Independent Lines seem to act in similar way.

(160) 797A/797A M2?670A/+:

(161) TABLE-US-00003 Pupae Adults Males:Females G 184 176 85:91 R + G 74 57 44:13

(162) 797C/797C M1?670A/+:

(163) TABLE-US-00004 Pupae Adults Males:Females G 169 157 89:68 R + G 94 67 54:13

(164) 797C/797C M2?670A/+:

(165) TABLE-US-00005 Pupae Adults Males:Females G 406 377 179:198 R + G 171 147 121:26

(166) 670A/+?797C/+M2:

(167) TABLE-US-00006 Pupae Adults Males:Females NF 198 192 92:100 G 162 147 67:80 R 149 72 43:29 R + G 45 22 20:2

(168) Average of all 3 lines: number of R+G females=21% of the number of R+G males, therefore substantial excess mortality in R+G females relative to males. This effect is not seen in R only or G only control females, nor in wild type.

Examples 13-15

(169) We have newly demonstrated: (5) sex-specific splicing in recombinant Aadsx-based minigene constructs; (6) sex-specific phenotype from a Cctra-based construct; and (7) sex-specific splicing in Aedes-Actin4-based constructs.

(170) At least some of each of these examples not only shows minigenes, but actually shows splicing to generate tTAV/tTAV2 or ubi-tTAV2.

Example 13: Aedes Doublesex (Dsx) Minigenes

(171) See also section entitled Aedes dsx Tra2 binding sites. We have isolated the Aedes aegypti dsx gene (Aadsx) and identified 6 transcripts from this region (FIG. 1). These are: 2 male-specific transcripts (M1 and M2), 3 female-specific transcripts (F1, F2 and F3) and a transcript found in both males and females (MF). We made two minigene constructs. In these constructs, the large majority of the intronic sequence was deleted. For example, DSX minigene1 is approximately 4.4 kb in length, whereas its terminal sequences are separated by approximately 26 kb in its natural context, i.e. in the genomic DNA of Aedes aegypti.

(172) The splicing in minigene2 of FIG. 1 is illustrative as splicing occurs in the female form in both males and females. This may mean that this system depends on alternative splice acceptor use. In this model, there is competition between alternative splice acceptors, with some sex-specific factor biasing this, the sex-specific factor probably being Tra. But deleting the M1 and M2 3 splice acceptors forces splicing in the F forms, by removing the alternative.

(173) Therefore, it is preferred that one or more of the female-specific (F1 and/or F2) 3 splice acceptors are provided together with an additional 3 splice acceptor. Most preferably, said additional splice acceptor is the 3 splice acceptor of M1 or M2 splice variant (or both), although it is envisaged that this is not essential as other known 3 splice acceptors are likely to function.

(174) FIG. 1 illustrates the various transcripts produced by alternative splicing of the Aedes aegypti doublesex gene (Aadsx). It will be appreciated that Aedes aegypti is also known as Stegomyia aegypti. The figure shows the Aadsx gene from the fourth exon, which is not alternatively spliced, i.e. is present in all transcripts discussed here. Numbering is from the first nucleotide of the fourth exon (acgacgaact, nucleotides 1-10 of SEQ ID NO:1, nucleotides 1316-1325 of SEQ ID NO:153). Note that the diagram is not to scalethe introns are much longer than the exons. The total alternatively spliced region comprises over 43 kb.

(175) This minigene fragment was included in an expression construct (LA3515). Transgenic Aedes aegypti were generated by site-specific recombination into an attP site, using the method of Nimmo et al (2006: Nimmo, D. D., Alphey, L. Meredith, J. M. and Eggleston, P (2006). High efficiency site-specific genetic engineering of the mosquito genome. Insect Molecular Biology, 15: 129-136).

(176) A second, smaller minigene was constructed similarly (DSX minigene2) and an expression construct for this was inserted into the same attP site as DSX minigene1, to allow direct comparison (LA3534). DSX minigene2 did not show sex-specific splicing. This indicates that sequences present in DSX minigene1 but not in DSX minigene2 (approx 2029 bp, see FIG. 1 and SEQ ID NO. 150, where exons are found at positions 29-163 and 1535-2572) are essential for correct alternative splicing, even though the first alternatively spliced intron, and the exonic sequence immediately flanking it, is present in both constructs.

(177) We have produced two transgenic lines (LA3491 and LA3534) using minigene constructs of Aedes aegypti dsx gene. LA3491 is a fusion of shared exon4, the female-specific cassette exons, and part of the first shared 3 exon (exon 5 in transcript M1).

(178) Transcripts from the minigene region of LA3491 were analysed by reverse transcriptase PCR (RT-PCR) and sequencing. Transcripts corresponding to alternative splicing in the F2 form were found in females but not in males (FIGS. 2 and 3) and in the F1 form there was some male expression but it was very low (FIG. 4). While transcripts corresponding to the M1 form were detected in males but not in females (FIG. 2). Since the minigene did not contain the 3 splice acceptor of the M2 variant, this transcript was not possible from this construct. This minigene does not contain any exogenous sequence, though it clearly demonstrates sex-specific splicing of an Aadsx fragment, indeed a highly deleted minigene fragment.

(179) It will be apparent that certain sequences are important for controlling splicing and should therefor be retained, as discussed elsewhere. This can be easily established by deletion of certain portions and testing for alternative splicing by RT-PCR for instance.

(180) FIG. 2 shows RT-PCR of males and females from LA3491 Aedes aegypti transgenic line using the primers 688ie1-transcr (SEQ ID NO. 4) and 790Aedsx-m-r2 (SEQ ID NO. 5). Using these primers, splicing in the F2 pattern would give a band of approximately 985 bp while splicing in the M1 pattern would give a band of approximately 516 bp. A band of approx 985 bp (F2) appeared only in lanes representing females and a band of approx 516 bp male specific transcript 1 (M1) appeared only in males. These bands have been sequenced and show that correct splicing had occurred, i.e. F2-type and M1-type respectively. The absence of bands in the no RT controls (-RT CON) shows that there was no genomic DNA contamination in the samples. Lanes 1 and 11 are Marker (SmartLadder? from Eurogentec, bands from 1.5 kb to 0.2 kb are indicated). Lanes 2 and 3 are negative controls (no reverse transcriptase) and lanes 2-9 represent reactions performed on extracts from males or females as marked.

(181) FIG. 3 shows RT-PCR of males and females from LA3491 Aedes aegypti transgenic lines using the primers 688ie1-transcr (SEQ ID NO. 4) and 761Aedsx-fem-r (SEQ ID NO. 6). Using these primers, splicing in the F2 pattern would give a band of approximately 525 bp. A band of approximately 525 bp was present in reactions on extracts from females, but not from corresponding reactions on extracts from males. Sequencing of this 525 bp band confirmed that correct, i.e. F2-type splicing had occurred. Marker (SmartLadder? from Eurogentec, bands from 1.5 kb to 0.2 kb are indicated).

(182) FIG. 4 shows RT-PCR of males and females from LA3491 Aedes aegypti transgenic lines using the primers 688ie1-transcr (SEQ ID NO. 4) and AedsxR1 (SEQ ID NO. 4). Using these primers splicing in the F1 pattern would give a band of 283 bp. A band of approximately 283 bp is present predominantly in females, although there is evidence of a small amount of splicing in males. Sequencing confirmed that this band did indeed correspond to splicing in the F1 pattern. Marker (SmartLadder? from Eurogentec, bands from 1.5 kb to 0.2 kb are indicated).

(183) LA3534 is identical to LA3491 except for a 3 deletion of approx 2 kb. This construct showed no differential splicing between male and females (FIG. 1, minigene 2). RT-PCR gels have not been shown for this case. Based on these results several constructs have been designed to incorporate the sex-specific splicing of LA3491 (FIG. 1, minigene 1) into a positive-feedback system. LA3612 (FIG. 5), which incorporates a fusion of ubiquitin and tTAV2 into the dsx coding region, is designed so that when the F2 female transcript is produced, the ubiquitin is cleaved and the tTAV2 is released to initiate and sustain the positive feedback system. LA3619 (FIG. 5) has tTAV2 without ubiquitin and using its own translation start codon. LA3646 (FIG. 5) is identical to LA3619 except the start codons for the dsx gene have been mutated; this should improve the quantity of tTAV2 produced by removing non-specific translation.

(184) FIG. 5 is a diagrammatic representation of plasmids based around the splicing in Aedes aegypti dsx minigene. For clarity it will be understood that the first female intron represents any of F1, F2 or F3 splicing, and tTAV in the diagram refers to tTAV2 (it will be appreciated that other proteins or other versions of tTA or tTAV could alternatively be used). In each of these plasmids, apart from LA3491, heterologous sequence has been added to the F2 exon. Putative ATG represents any ATG triplet sequence in exonic sequence located 5 relative to the heterologous DNA. In LA3646 these putative translation start codons (putative ATG) were removed or modified. In the case of construct LA3612, translation from an upstream (5) ATG that is in frame with the ubi-tTAV coding region will still (assuming no intervening stop codon) produce functional tTAV, following separation of the ubiquitin and tTAV moieties by protease action. The various alternative splicing cassettes are operably linked to a suitable promoter, transcriptional terminator and other regulatory sequences.

(185) This example shows sex-specific splicing of a highly compressed minigene fragment in a heterologous context (i.e. heterologous promoter, 5 UTR and 3UTR). Although it does not show differential expression of a non-Aedes sequence, as the alternatively spliced exons are derived from the Aadsx gene and do not contain additional material, it does clearly illustrate the feasibility of this approach. In any case, the promoter, 5 UTR and 3UTR are heterologous. We have additional constructs which illustrate several different methods for obtaining differential (sex-specific) expression of a heterologous protein by this dsx.

(186) TRA Sequence Alignment

(187) Pane et al. (2002) suggested that certain sequences related to the known binding sites of the Tra/Tra-2 complex in Drosophila might be important in regulating the splicing of Cctra, and this also known for Drosophila dsx and has also been suggested for Anopheles gambiae dsx (Scali et al 2005). The consensus sequence is variously described as UC(U/A)(U/A)C(A/G)AUCAACA (Pane et al), SEQ ID NO. 8, or UC(U/A)(U/A)CAAUCAACA (Scali et al 2005), SEQ ID NO. 9.

(188) It is noteworthy that these definitions are extremely similar. Pane et al identify 8 partial matches to this consensus in the Cctra sequence (7 or more nucleotides matching the 13 nucleotide consensus sequence. Scali et al identify 6 matches in Agdsx (9/13 or better). Such sequences are also known to regulate the alternative splicing of the Drosophila gene fruitless; Scali et al review 3 matches in that sequence (12/13 or better). Correct splicing of dsx may also require a purine-rich region, as discussed by Scali et al.

(189) As can be seen from the Table 2 and FIG. 7, we have identified what are thought to be significant clusters of binding sites for Tra/Tra2 in our Aedes aegypti dsx minigene1.

(190) Moth Dsx Sequence Alignment and Conserved Motifs

(191) FIG. 6A and FIG. 6B show an alignment of the second female-specific exons and flanking sequences of dsx genes from pink bollworm (Pectinophora gossypiella, PBW-dsx, SEQ ID NO. 146), silk worm (Bombyx mori, bombyx-dsx, SEQ ID NO. 147) and codling moth (Cydia pomonella, codling-dsx, SEQ ID NO. 148). The second female-specific exon is shown in bold. We identified multiple copies of a short, repeated nucleotide sequence, conserved in sequence and approximate location between these relatively distantly related moths; these are located just 5 to the female-specific exon. The conserved repeats AGTGAC/T are underlined. Asterisks (*) represent identical nucleotides, dashes (-) represent gaps for best alignment. The exons are represented in the SEQ ID NOS. by the following nucleotide numbering: SEQ ID NO. 146 289-439; SEQ ID NO. 147 339-492; and SEQ ID NO. 148 285-439.

(192) Aedes Dsx Tra2 Binding Sites.

(193) In females of Drosophila melanogaster, Tra and a product from the constitutively active gene tra2, act as splicing regulators by binding to splice enhancer sites on the pre-mRNA of dsx, which activates the weak 3 acceptor site of the female-specific exon (Scali et al). In males there is no expression of TRA and the weak 3 acceptor site is not recognised and splicing occurs at the male exon. To look for putative Tra/Tra2 binding sites we used the consensus sequence of these binding sites deduced for Drosophila Tra/Tra2 and looked for the distribution of these in the Aedes aegypti dsx gene sequence. This is shown in Table 2, below.

(194) TABLE-US-00007 TABLE2 Sequence Present Identity Identity SEQ w= TorA in with with ID Name r= AorG Minigene1 Position consensus wwcrat NO. Consensus tcwwcratcaaca / / /13 /6 138 1 tcaacaagcaaca Y 14917 12 5 10 2 ttatcaaacaaca Y 364 11 5 11 3 tcatcaattaaaa 1015 11 6 12 4 tcatcaatcaaac 6502 11 6 13 5 tcttcaaccaacc Y 14958 11 5 14 6 cctacaatctaca Y 14973 11 6 15 7 tcttagatcaaaa 16553 11 5 16 8 tcttcgatcatta 17386 11 6 17 9 ccaacaatctaca 28802 11 6 18 10 tcaaagatcacca 42096 11 5 19 11 tcttcggtcgacg Y 256 11 5 20 12 tcgacaaacaaaa 1277 11 <5 21 13 tattcaaacaacg 4061 11 5 22 14 ttttcgataaaaa 4380 10 6 23 15 tcttcagtctgca 5399 10 5 24 16 gattcaatcatca 7723 10 6 25 17 ttatcgagcaaaa 8137 10 5 26 18 tcataactcaaga 9062 10 <5 27 19 tcagaaatcaaaa 9126 10 <5 28 20 tctttaatttaca 10639 10 5 29 21 tttacaatcctca 10646 10 6 30 22 tcatagatcagga 11214 10 5 31 23 acctcaaacaaca 11989 10 <5 32 24 tcatcgaacaccc 12020 10 5 33 25 tcaataatcgtca 12199 10 5 107 26 tcatcaaacgtca 13287 10 5 108 27 ttatcgttaaaca Y 13439 10 5 109 28 taaacagtcaata Y 13446 10 5 110 29 tacacgatcagca Y 14096 10 5 111 30 aatacaaacaaca Y 14637 10 5 112 31 tcatcaacaagca Y 14914 10 5 113 32 tctacaaaccaga Y 14980 10 5 114 33 acatcgattcaca 16085 10 6 115 34 cgctcaatcaaca 16175 10 5 116 35 tctaccataaaaa 16511 10 5 117 36 aaatgaatcaaca 20044 10 5 118 37 acatcgttcaacg 21374 10 5 119 38 tcttgattcacca 21580 10 <5 120 39 tctgcagacaaca 22408 10 <5 121 40 tcttcggtaatca 23285 10 5 122 41 tctataaacaata Y 25436 10 <5 123 42 taaacaataaata Y 25440 10 6 124 43 taaacaagcaaaa 28242 10 5 125 44 tcaacgatcggcg 30309 10 6 126 45 tgatccatcatca 30910 10 5 127 46 tcaacatgcaaga 32295 10 <5 128 47 tcttaaataaaga 32862 10 5 129 48 tcaaagatctata 40551 10 5 130 49 taatgaattaaca 40847 10 5 131 50 tttaccatcaact 41712 10 5 132 51 taatgaaacaaca 43380 10 <5 133 52* gtttcaattaaaa Y 13500 9 6 134 53* tattcaattataa Y 13602 9 6 135 54* tcttcaatcgttt Y 15002 9 6 136 55* tcaacgatccttt Y 15533 9 6 137 * = in 3491, only 9/13 but 6/6 in core. This table does not include 9/13 identities apart from the ones that are in 3491 with 6/6 identity with core sequence of wwcrat. This consensus core sequence (WWCRAT) is particularly preferred.

(195) FIG. 7 is a diagrammatic representation of putative Tra/Tra2 binding sites within the dsx coding region of plasmid LA3491. This diagram is approximately to scale and represents a sequence of approximately 4 kb. We can calculate the chance of a random match to the Tra/Tra2 consensus sequence. Assuming all 4 nucleotides occur at equal frequency, the chances of any given nucleotide in a random sequence being the first nucleotide of a 10/13 or better match to the consensus is approx 7?10.sup.?4. Therefore, one would expect slightly less than one such match per 1000 nucleotides of such random sequence. The calculation for this is below:

(196) Sex-Specific Splicing: Probabilities

(197) Questions

(198) A binding site consensus sequence consists of 13 bases. Ten of those (fixed) positions (call this set X) must each be one specific base. The other three (call this set Y) can each be one of two specific bases. Assuming that each possible base A, G, C and T is equally likely and that the base at each position is independent of the bases at the other positions, what is the probability of a 13-base sequence selected at random exactly matching this sequence? What are the probabilities of such a sequence being a near mismatch (allowing for up to one, two, three or four differences)? The answers are provided in Table 2 below and the workings are shown thereafter.

(199) Answers

(200) TABLE-US-00008 TABLE 3 No. of positions Probability Probability mismatched (fraction) (to 3 d.p.) none, i.e. exact match $\frac{1}{2^{23}}$ 1.192 ? 10.sup.?7 up to 1, i.e. at least 12 positions match $\frac{17}{2^{22}}$ 4.053 ? 10.sup.?6 up to 2, i.e. at least 11 positions match $\frac{133}{2^{21}}$ 6.342 ? 10.sup.?5 up to 3, i.e. at least 10 positions match $\frac{23}{2^{15}}$ 7.019 ? 10.sup.?4 up to 4, i.e. at least 9 positions match $\frac{33863}{2^{23}}$ 4.037 ? 10.sup.?3

(201) Workings:

(202) $P\left(\mathrm{exact}\mathrm{match}\right)=P_0={\left(\frac{1}{4}\right)}^{10}{\left(\frac{1}{2}\right)}^3=\frac{1}{4^{10}?2^3}=\frac{1}{2^{23}}=1.192?{10}^{-7}\mathrm{to}3d.p.\left(3d.p.\mathrm{all}\mathrm{below}\right)$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{exactly}1\mathrm{position}\right)=P\left(\mathrm{mismatch}\mathrm{at}\mathrm{one}\mathrm{of}\mathrm{the}10X\mathrm{positions}\mathrm{or}\mathrm{mismatch}\mathrm{at}\mathrm{one}\mathrm{of}\mathrm{the}3Y\mathrm{positions}\right)=P_1=10{\left(\frac{1}{4}\right)}^9\left(\frac{3}{4}\right){\left(\frac{1}{2}\right)}^3+3{\left(\frac{1}{4}\right)}^{10}{\left(\frac{1}{2}\right)}^3=\frac{\left(10?3\right)+3}{4^{10}?2^3}=\frac{33}{2^{23}}=3.934?{10}^{-6}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{exactly}2\mathrm{positions}\right)=P\left(\mathrm{mismatches}\mathrm{at}2\mathrm{of}\mathrm{the}10X\mathrm{or}\mathrm{mismatch}\mathrm{at}1\mathrm{of}\mathrm{the}10X\mathrm{and}1\mathrm{of}\mathrm{the}3Y\mathrm{or}\mathrm{mismatches}\mathrm{at}2\mathrm{of}\mathrm{the}3Y\right)=P_2=\frac{10!}{2!8!}{\left(\frac{1}{4}\right)}^8{\left(\frac{3}{4}\right)}^2{\left(\frac{1}{2}\right)}^3+10?3{\left(\frac{1}{4}\right)}^9\left(\frac{3}{4}\right){\left(\frac{1}{2}\right)}^3+3{\left(\frac{1}{4}\right)}^{10}{\left(\frac{1}{2}\right)}^3=\frac{\left(\left(45?3^2\right)+\left(30?3\right)+3\right)}{2^{23}}=\frac{498}{2^{23}}=\frac{249}{2^{22}}=5.937?{10}^{-5}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{exactly}3\mathrm{positions}\right)=P\left(\mathrm{mismatches}\mathrm{at}3\mathrm{of}\mathrm{the}10X\mathrm{or}\mathrm{mismatches}\mathrm{at}2\mathrm{of}\mathrm{the}10X\mathrm{and}1\mathrm{of}\mathrm{the}3Y\mathrm{or}\mathrm{mismatches}\mathrm{at}1\mathrm{of}\mathrm{the}10X\mathrm{and}2\mathrm{of}\mathrm{the}3Y\mathrm{or}\mathrm{mismatches}\mathrm{at}3\mathrm{of}\mathrm{the}3Y\right)=P_3=\frac{10!}{3!7!}{\left(\frac{1}{4}\right)}^7{\left(\frac{3}{4}\right)}^3{\left(\frac{1}{2}\right)}^3+\frac{10!}{2!8!}3{\left(\frac{1}{4}\right)}^8{\left(\frac{3}{4}\right)}^2{\left(\frac{1}{2}\right)}^3+10?3{\left(\frac{1}{4}\right)}^9\left(\frac{3}{4}\right){\left(\frac{1}{2}\right)}^3+{\left(\frac{1}{4}\right)}^{10}{\left(\frac{1}{2}\right)}^3=\frac{\left(\left(120?3^3\right)+\left(45?3^3\right)+\left(30?3\right)+1\right)}{2^{23}}=\frac{5356}{2^{23}}=\frac{1339}{2^{21}}=6.385?{10}^{-4}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{exactly}4\mathrm{positions}\right)=P\left(\mathrm{mismatches}\mathrm{at}4\mathrm{of}\mathrm{the}10X\mathrm{or}\mathrm{mismatches}\mathrm{at}3\mathrm{of}\mathrm{the}10X\mathrm{and}1\mathrm{of}\mathrm{the}3Y\mathrm{or}\mathrm{mismatches}\mathrm{at}2\mathrm{of}\mathrm{the}10X\mathrm{and}2\mathrm{of}\mathrm{the}3Y\mathrm{or}\mathrm{mismatches}\mathrm{at}1\mathrm{of}\mathrm{the}10X\mathrm{and}3\mathrm{of}\mathrm{the}3Y\right)=P_4=\frac{10!}{4!6!}{\left(\frac{1}{4}\right)}^6{\left(\frac{3}{4}\right)}^4{\left(\frac{1}{2}\right)}^3+\frac{10!}{3!7!}3{\left(\frac{1}{4}\right)}^7{\left(\frac{3}{4}\right)}^3{\left(\frac{1}{2}\right)}^3+\frac{10!}{2!8!}3{\left(\frac{1}{4}\right)}^8{\left(\frac{3}{4}\right)}^2{\left(\frac{1}{2}\right)}^3+10{\left(\frac{1}{4}\right)}^9\left(\frac{3}{4}\right){\left(\frac{1}{2}\right)}^3=\frac{\left(\left(210?3^4\right)+\left(120?3^4\right)+\left(45?3^3\right)+\left(10?3\right)\right)}{2^{23}}=\frac{27975}{2^{23}}=3.335?{10}^{-3}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{up}\mathrm{to}1\mathrm{position}\right)=P_0+P_1=\frac{1+33}{2^{23}}=\frac{17}{2^{22}}=4.053?{10}^{-6}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{up}\mathrm{to}2\mathrm{positions}\right)=P_0+P_1+P_2=\frac{1+33+498}{2^{23}}=\frac{532}{2^{23}}=\frac{133}{2^{21}}=6.342?{10}^{-5}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{up}\mathrm{to}3\mathrm{positions}\right)=P_0+P_1+P_2+P_3=\frac{1+33+498+5356}{2^{23}}=\frac{5888}{2^{23}}=\frac{23}{2^{15}}=7.019?{10}^{-4}$$P\left(\mathrm{mismatch}\mathrm{in}\mathrm{up}\mathrm{to}4\mathrm{positions}\right)=P_0+P_1+P_2+P_3+P_4=\frac{1+33+498+5356+27975}{2^{23}}=\frac{33863}{2^{23}}=4.037?{10}^{-3}$

Experiment 14: Cctra

(203) We have one line of LA3097 (LA3097A) which shows very good expression of its fluorescent marker; it is unknown if this line is a single integration event. This line does show evidence of sex-specific splicing, when reared off tetracycline all the females die as embryos, and when it is on 30 ?g/ml of tetracycline both males and females survive.

(204) This example is important. It shows that Cctra provides sex-specific alternative splicing in Aedes, and that this can be used to give sex-specific lethality. This, therefore, provides evidence of the phylogenetic range for Cctra splicing. Thus, it is entirely plausible that the present invention can be applied to all Diptera, as we have shown that Cctra works in Drosophila, tephritids and mosquitoes, which essentially spans the whole Dipteran Order.

(205) It is surprising that Cctra works in Aedes, given the rapid sequence evolution of tra.

(206) We transformed Aedes aegypti with construct LA3097. Heterozygous males from the resultant transgenic line were crossed to wild type and the progeny reared in aqueous medium supplemented with tetracycline to a final concentration of 30 ?g/ml. Adults were recovered as follows: 14 males and one female, thus showing significant female-specific lethality.

(207) This species and strain normally has a sex ratio of approximately 1:1, therefore this construct gave female-specific lethality in Aedes aegypti. Equivalent constructs which did not contain the Cctra intronic sequence gave non-sex-specific lethality. Therefore, the Cctra intron can be used to provide differential (i.e. sex-specific) regulation of gene expression in mosquitoes, and this can further be used to provide sex-specific lethality and a method for the selective elimination of females from a population.

(208) In more detail: on 0 ?g/ml tetracycline, males survive only to pupae, i.e. don't make it to adult. Females die so early that we don't see them, probably as embryos, so there is still a differential effect between the sexes. However, the pupal lethality in males suggests that the system is not completely switched off in males. The single insertion line that we recovered is unusual, in that it shows extremely strong expression of the marker; other insertions with more typical expression levels might well not show male lethality.

(209) Splicing in LA3097A

(210) Analysis of splicing of LA3097 from LA3097A transgenic mosquitoes by RT-PCR showed that males and females shared two transcripts, an approximately 950 bp band and a fainter band of approximately 800 bp (FIG. 59). Sequencing of these bands showed that the ?900 bp band corresponds to a non-sex-specific splice variant (AeM2, 920 bp), and the fainter band was a mixture of a non-sex-specific splice variant (AeM1, 804 bp) and the female form (AeF1, ?765 bp), see FIG. 60. The splicing of the AeF1 transcript was identical to that shown for this construct in Medfly (FIG. 33). The splicing of the M transcripts differs somewhat from that seen in the native context (Cctra splicing in Medfly, either the native gene or as we observed from LA3097 in transgenic Medfly); in AeM1 the second alternatively spliced exon (ME1b) is not included in the mature AeM1 transcript and in AeM2 the second alternatively spliced exon (ME2b) is similarly not included in the mature AeM2 transcript. In other words, for each of these transcripts the first but not the second cassette exon is present, relative to the Medfly prototype. Note that, as a consequence of the absence of the second cassette exon in AeM1, and the reading frame of tTAV2 relative to the first cassette exon in this construct, splicing in the AeM1 pattern does not lead to interruption of the tTAV2 open reading frame, but rather to the addition of 39 nucleotides (corresponding to 13 amino acids) between the ATG and the rest of the tTAV2 open reading frame. It is likely that this variant of tTAV2 may retain some activity, relative to normal or prototypic tTAV2 (as encoded by the F1 splice variant). In the absence of tetracycline, a phenotypic effect was observed in males as well as in females, though weaker in males than females. Production of a partially active variant of tTAV2 from the AeM1 transcript in males (and females) may explain this.

(211) FIG. 59shows RT-PCR of males and females from LA3097A Aedes aegypti transgenic line using the primers HSP (SEQ ID NO. 139) and VP16 (SEQ ID NO. 140). Using these primers, splicing in the CcF1 pattern (i.e. corresponding to the F1 variant of Ceratitis capitata) would give a band of approximately 765 bp and splicing in the CcM1 1005 bp and CcM2 1094 bp. In both males and females, a strong band of approximately 950 bp (1) was observed along with a fainter band of approximately 800 bp (2). Marker (SmartLadder? from Eurogentec, bands from 1.5 kb to 0.4 kb are indicated).

(212) Sequence analysis of several clones from band 2 (i.e. AeM1/AeF1 splice variants) from males and females showed that one of five clones from females showed AeM2 splicing (20%), whereas in males three of the four clones showed AeM2 splicing (75%); all the other clones showed AeF1 splicing. This indicates that there is more AeF1 transcript present in females than in males and this would explain the differential killing effect seen between them.

(213) FIG. 60 Illustrates the various transcripts produced by alternative splicing of Cctra from LA3097A Aedes aegypti transgenic line. 3097 represents the DNA sequence of Cctra and the numbers relate to figure described elsewhere. Shading and boxes also relate to FIG. 33. Note that the diagram is not to scale.

Example 15: Aedes Actin-4

(214) We have eleven lines of LA3545, which uses the Aedes actin-4 gene (AeAct-4 or AaAct4) to drive expression of tTAV2. In construct LA3545, a sequence encoding tTAV2 has been inserted into the second exon of AaAct4 (FIG. 10). For transcripts spliced in the pattern characteristic of AaAct4 splicing in females, the ATG of the tTAV2 coding region will be the first (5-most) ATG of the transcript. Splicing in the pattern characteristic of AaAct4 splicing in males introduces an array of start and stop codons before the tTAV2 sequence which tends to inhibit or interfere with translation from the ATG of the tTAV2 coding region. These lines should only express tTAV2 in female pupae. The splicing is shown in FIG. 8, below.

(215) FIG. 8 shows RT-PCR of male and female adults from LA3545AeC Aedes aegypti transgenic line using the primers Agexon1F (SEQ ID NO. 141) and TETRR1 (SEQ ID NO. 142). Using these primers, splicing in a pattern equivalent to that of the native AaAct4 gene would give bands of approx 347 bp for the female-type splice variant and of approx 595 bp for the male-type splice variant. A band of approx 347 bp band (F) was found only in reactions on extracts from females; a band of approx 595 bp (M) was found in both males and females. Sequencing has confirmed that the correct splicing occurred in males and females. Marker (SmartLadder? from Eurogentec, bands from 1.5 kb to 0.2 kb are indicated).

(216) We also have transgenic Aedes aegypti carrying construct LA3604, which is similar to LA3545 except it has an engineered start codon in the portion of exon 1 that is present in both male-type and female-type transcripts (FIG. 10). This is arranged to be the first ATG in either transcript type. LA3604 encodes tTAV2 fused to ubiquitin (LA3545 codes tTAV, while LA3604 codes ubi-tTAV2). This construct should produce a fully functional tTAV2 protein in females only, even if the male form is expressed in females the extra male exon contains several start and stop codons that would prevent translation of the Ubi-tTAV2 fusion protein.

(217) The alternative splicing of AaAct4 occurs in the 5 UTR (of the native gene). It may or may not have a regulatory role in the native gene. One possibility is as follows: in the female-specific splice variant, the start codon of the AaAct4 coding region is the first ATG of the transcript. However, in the male-specific splice variant there are several additional ATG sequences 5 to the start codon of the AaAct4 coding region; most of these have in-frame stop codons a short distance 3. This sequence arrangement may interfere with the efficient translation of the AaAct4 protein and thereby reduce expression of the protein in males as compared with females. This is the arrangement in LA3545.

(218) However, a greater differential effect between males and females would be expected if the intron was included in coding region (rather than 5 UTR), i.e. inserted between the start and stop codons of the polynucleotide for expression in the organism. In this case, the male-specific cassette exon would change the coding potential of the transcript, rather than simply interfering with translation.

(219) This is achieved in construct LA3604. We modified the shared first exon to include an ATG sequence in a suitable sequence context for translational initiation. In this modified sequence, this is the first ATG in either the male-type (M) or female-type (F) splice variants. Following splicing in the F form, this (engineered) 5 ATG is in frame with the ubi-tTAV coding region. F-type transcripts would therefore encode a fusion protein, comprising sections encoded by (i) part of what is normally Act4 5 UTR (but here obviously translated, and so not UTR at all), (ii) ubiquitin coding region and (iii) tTAV2 coding region.

(220) Activity of cellular ubiquitin proteases will release the tTAV2 protein. Translation from the engineered 5 ATG would be terminated by in-frame stop codons in the additional sequence (cassette exon) present in transcripts spliced in the M form. This would therefore prevent expression of functional tTAV2 in males, thereby giving sex-specific expression of tTAV2. Obviously, this gives a general method for sex-specific expression of a protein, by replacing the tTAV2 segment with another protein or sequence of interest. Using this strategy we have provided transgenics and shown sex-specific splicing (FIG. 9).

(221) FIG. 9 shows RT-PCR of males and females from LA3604AeA Aedes aegypti transgenic line using the primers Agexon1F (SEQ ID NO. 141) and TETRR1 (SEQ ID NO. 142). Using these primers, splicing in the female form would give a band of approximately 575 bp, while inclusion of the male-specific cassette exon would increase this to approximately 823 bp. A band of approx 575 bp was seen from each female analyzed, while a band of approx 823 bp was seen from each male analyzed. These bands appear to be substantially specific to the respective sexes. Sequencing of these bands showed the correct splicing had occurred in males and females. Marker: SmartLadder? from Eurogentec, bands from 1.5 kb to 0.2 kb are indicated.

(222) FIG. 10, below, is a diagrammatic representation of plasmids LA3545 and LA3604. S1: shared exon 1; M1: additional sequence included in male-specific exon 1; S2: shared exon 2 (5 end only); ubi: sequence encoding ubiquitin; tTAV2: sequence encoding tTAV2.

(223) In several of the LA3545 transgenic lines a sex- and tissue-specific effect was observed: females are flightless. Two of the lines show a 90-100% female flightless phenotype one line shows 70% flightless and another 50%. This phenotype is presumably due to female-specific expression of tTAV2 in the developing flight muscles. The difference in the phenotypes between the lines is due to positional effects on the expression of the AaAct4 promoter. Due to a genes position in the genome expression can be influenced by a number of factors (heterochromatic or euchromatin regions, enhancer and suppressor elements, proximity to other genes) which can be seen readily in the fluorescent markers used to identify transgenics. All eleven lines of LA3545 were identified because they have different fluorescent profiles, even though they have the same promoters and marker. This variation is due to positional effects. This would then mean that we would expect some lines of LA3545 to express more tTAV2 than other because of positional effects, and those lines that do express more would give a female-specific flightless phenotype.

(224) To test this hypothesis we developed a separate Aedes aegypti line with a tetO-DsRed2 reporter gene (LA3576 see FIG. 17 and SEQ ID NO. 143), when crossed with the different LA3545 lines this would allow the visualisation of where and when the Actin4-tTAV2 was expressing. Out of 8 LA3545 lines crossed to LA3576 all showed female-specific indirect flight muscle fluorescence in late L4 larvae, pupae and adults. In four of the lines DsRed2 expression appeared to be specific (i.e. exclusive) to the female indirect flight muscles; in the other four additional tissues showed expression of DsRed2. This phenomenon, where expression of a transgene depends in part on the region or point in the genome into which it has inserted, is called position effect, and will be well known and understood by the person skilled in the art.

(225) Using LA3576 proved that the expression of tTAV2 in LA3604 was female-specific, occurs mainly in the indirect flight muscles and is stage-specific. Several different tetO-effector constructs were then constructed to analyse their effects. The tetO-MichelobX transgenics (LA3582, see FIG. 15 and SEQ ID NO. 144) when crossed to LA3545 all showed female-specific flightless phenotypes that could be repressed by tetracycline. This proves that Actin4 can be used to drive an effector gene in a stage, tissue and sex-specific manner.

(226) Because some lines of LA3545 had a female-specific flightless phenotype without the presence of an induced effector gene, this showed that tTAV2 could act as an effector molecule. tTAV2 is composed of a tTA, a tetO binding domain and VP16, a herpes simplex virus protein. VP16 activates transcription of immediate early viral genes by using its amino-terminal sequences to attach to one or more host-encoded proteins that recognise DNA sequences in their promoters. In LA3604 a tetO-VP16 effector gene has been added to enhance the effect of tTAV2. In three transgenic lines of LA3604 this has caused a 100% female-specific flightless phenotype when reared without tetracycline, showing that VP16 is an effective effector molecule. Note that LA3604 has a potential start codon (ATG) engineered 5 to the alternatively spliced intron. Therefore, in this construct, the male-specific exon is expected to interrupt the open reading frame encoding tTAV (ubi-tTAV); since the male-specific sequence contains several stop codons, this will tend to reduce or eliminate production of functional tTAV in males. By way of comparison, the male-specific exon is 5 to the start codon of tTAV in LA3545. However, by inserting a number of start codons 5 to the start codon of tTAV (which is the first ATG of the female transcript but not of the male transcript), none of these additional start codons being suitable for efficient production of functional tTAV due to being out of frame or having intervening stop codons, this arrangement will also tend to reduce or eliminate production of functional tTAV in males, consistent with the phenotypic data above.

Example 16: Use of Ubiquitin and Intron Positioning

(227) We have newly made Cctra-based constructs with the Cctra intron cassette in a variety of different contexts, i.e. flanked by different sequences. Various lines of transgenic Medfly carrying these have been constructed. This shows that the system is general and robust, i.e. that it will work for a wide range of heterologous sequences of interest.

(228) We also have at least one newly made example of a Cctra-ubi-tTAV fusion giving correct splicing (DsRed-cctra-ubi-tTAV).

(229) Preferred examples of the functional protein place the coding sequence for either ubiquitin or tTA, or their functional mutants and or variants such as tTAV, tTAV2 or tTAV3, 3 to the intron. These are arranged so that these elements are substantially adjacent to the 3 end of the intron, more preferably such that the coding region starts within 20 nucleotides or less of the 3 intron boundary), and most preferably, immediately adjacent the 3 end of the intron, although this is less relevant if the Ubiquitin system is used.

(230) Preferred examples of constructs according to the present invention are listed in Table 4, below. It will be appreciated that LA1188 is not within the scope of the present invention, as it does not encode a functional protein, i.e. it doesn't work properly. This is thought to be because of the unexpected use of a splice donor 4 bp 5 to the junction with Cctra intron sequence, leading to a frameshift that is induced in all splices. It is, therefore, included for the sake of information only.

(231) TABLE-US-00009 TABLE 4 Construct NO. Species tra intron position from tra intron (FIGS #.) is from ATG (bp) is fused to- LA1188 (80) Medfly +132 tTAV LA3014 (29) Medfly +22 ubiquitin LA3166 (30) Medfly +136 ubiquitin LA3097 (27) Medfly +0 tTAV LA3077 (26) Medfly +61 tTAV LA3233 (28) Medfly +0 tTAV2 LA3376 (31) Medfly +0 tTAV2 LA3376 (31) B. zonata +3 reaper KR LA3376 (31) B. zonata +0 tTAV3 LA3242 (32) C. rosa +3 reaperKR LA1038 (14) Medfly +21 Nipp1 (nipper) LA3054 (61) Medfly +811 DsRed-ubiquitin LA3056 (62) Medfly +811 DsRed-ubiquitin LA3488 (63) Medfly +949 Ubiquitin LA3596 (67) Medfly +949 Ubiquitin

(232) Table 4 shows constructs which contain a splice control sequence which is derived from a tra intron. The introns were derived from C. capitata (Medfly), B. zonata or C. rosa (see column 2). Said intron was inserted within the coding region such that the distance between the putative initiator ATG and the last nucleotide of the exon immediately preceding the tra intron was as should be indicated in column 3. Intron is inserted into or adjacent to coding region for either ubiquitin, tTAV, reaper.sup.KR, nipper or ubiquitin-DsRed as shown in column 4 These were generated and shown to successfully splice, by RT-PCR or phenotypically in Medfly and, in some cases, also either in Drosophila melanogaster (LA3077) or Anastrepha ludens (LA3097, LA3233, LA3376). In addition, the distance between the ATG and the end of the exon immediately preceding the tra intron (assuming splicing in F1-like form) can range from 0 bp to at least +949 bp without adverse consequences to splicing (see Table 4, column 3). Thus, it is reasonable to assume that this distance can be up to at least 900 and preferably up to at least 949 bp.

(233) Further information on these examples is summarized in Table 5. The preferred option is to use no endogenous sequence to achieve correct alternative splicing control of expression (+0 bp in table 4). We prefer to insert the tra intron between the flanking dinucleotides TG . . . GT in the coding region of the protein of interest to be alternatively spliced to ensure correct splicing as this may be important, however we will not restrict ourselves to this if necessary as other flanking nucleotides may function correctly as well. Examples LA1038, LA3054 and LA3056 include some endogenous flanking exonic sequence from the natural Cctra gene. In Table 5, if 6 nucleotides or less (including the ATG start codon) are included of particular fusions to the 3 or 5 of the splice junction, for the summary purposes of this table these will not be considered to be part of the fusion. Table 4 can be correlated with table 3 to find which tra intron (Cctra, Bztra or Crtra) is used in each example. Again, LA1188 is included only for the purposes of information and falls outside the present invention.

(234) TABLE-US-00010 TABLE 5 exonic tra exonic tra sequence sequence Construct NO. tra intron is tra intron is fused to 5 fused to 3 (FIGS #.) fused to 5 fused to 3 (bp) (bp) LA1188 Hsp70-tTAV tTAV +0 bp +0 bp (80) LA3014 Hsp70- ubiquitin-reap- +0 bp +0 bp (29) ubiquitin erKR-sv40 LA3166 Hsp70- ubiquitin-reap- +0 bp +0 bp (30) ubiquitin- erKR-sv40 LA3097 Hsp70 tTAV-K10 +0 bp +0 bp (27) LA3077 Hsp70-tTAV tTAV-K10 +0 bp +0 bp (26) LA3233 Hsp70 tTAV2-K10 +0 bp +0 bp (28) LA3376 Hsp70 tTAV2-K10 +0 bp +0 bp (31) LA3376 Sry-a tTAV3-sv40 +0 bp +0 bp (31) LA3376 HB reaperKR-sv40 +0 bp +0 bp (31) LA3242 HB reaperKR-sv40 +0 bp +0 bp (32) LA1038 Hsp70-tra Tra-Nipp1 +22 bp +20 bp (14) (nipper)-sv40 LA3054 Opie2-nls- tra-ubiquitin- +22 bp +20 bp (61) DsRed-tra tTAV-sv40 LA3056 Opie2-nls- tra-ubiquitin- +22 bp +242 bp (62) DsRed-tra tTAV-sv40 LA3488 Ie1-nls- ubiquitin-nls- +0 bp +0 bp (63) TurboGreen- DsRed-nls-sv40 nls-ubiquitin LA3596 Ie1-nls- ubiquitin-nls- +0 bp +0 bp (67) TurboGreen- DsRed-nls-sv40 nls-ubiquitin

(235) As mentioned above when an intron is placed 5 to a protein coding region (ORF-X), it is preferred to position or use ubiquitin 3 to the intron, 5 to ORF-X, thus and providing female-specific regulation of ORF-X, whilst introducing physical separation between that sequence and the tra intron, thereby reducing the chance that sequences within ORF-X will interfere with the splicing of the tra intron.

(236) Composite constructs and sequences are also envisaged, for example of the form: X-ubi-Y
with the alternatively spliced intron inserted between coding region X and the region encoding ubiquitin (ubi), or within the ubiquitin coding region, or between the region encoding ubiquitin and coding region Y. Thus X will be expressed irrespective of the splicing of the intron, while Y will only be expressed when the intron is spliced in a suitable form. Further configurations and arrangements of this general type will be apparent to the person skilled in the art. Some examples of this are LA3014, LA3054, LA3056, LA3166, LA3488 and LA3596 which all use ubiquitin fusions in this way demonstrating the ability of this idea to be successfully applied in transgenic Medfly. Alternative examples in transgenic mosquitoes include LA3604 and LA3612, showing the wide phylogenetic applicability of this system in not only different species (mosquitoes and Medfly), but also in different contexts including AaActin4, Aadsx and Cctra.

(237) LA3596 (see FIG. 67 and SEQ ID NO. 145) is of similar design to LA3488, intended to generate green fluorescence (by expression of nuclear localised TurboGreen fluorescent protein) in both sexes, but red fluorescence only in females (by expression of nuclear localised DsRed2 fluorescent protein). This is accomplished by the fusion of these two proteins, driven by the Hr5-Ie1 enhancer/promoter cassette, linked together with a short 11 amino acid linker (SG4 linker) and a coding region comprising ubiquitin (with one intended point mutation to stabilize the resulting protein by reducing its propensity to ubiquitin-mediated degradation) and the Cctra intron to limit DsRed2 expression to females. Transgenic Medfly were generated with this construct. Red fluorescence was limited to females in this line as expected, while green fluorescence was observed in all males and females. This could be used for sex separation by fluorescence screening for a particular fluorescent protein, in this case red fluorescence representing expression of DsRed2.

Example 17: Further Cctra Exemplification

(238) Reference is also made to LA3014 and LA3166 and phenotypic data therefrom in other Examples.

(239) We have previously made, and have obtained transgenics with, the Cctra intron in a functional protein other than tTAV, see LA3014 and LA3166. LA3014 contains a ubiquitin-reaper.sup.KR fusion downstream of a Cctra intron. Phenotypic data shows that LA3014 transgenic Medfly gave repressible female-specific lethality. RT-PCR analysis on RNA extracted from adult males and females raised off tetracycline, using primers and ReaperKR, demonstrate that correct splicing was occurring in females (508 bp band) and no such band was found in males (FIG. 37). LA3166 is another construct with the Cctra intron placed inside the ubiquitin coding region fused to reaper.sup.KR, but placed in a different position in ubiquitin. LA3166 also produces a dominant repressible female-specific lethal effect in Medfly.

(240) LA1038 is a new example of the use of the Cctra intron in a different sequence context, here placed in a fragment of Nipp1Dm called nipper that also splices correctly in transgenic Medfly when analysed by RT-PCR (FIG. 12). LA670 was required as a source of tTAV to drive expression of the alternatively spliced nipper.

(241) We have also newly made, and have obtained transgenics with, intron-only Cctra-based constructs with the intron in a different gene (many of the above examples, unless otherwise apparent, are in tTAV or one of its variants, i.e. tTAV2 or tTAV3). These constructs work as predicted. This is an important result, thus showing that there are not essential exonic sequences in Cctra that we have simply duplicated (in function, if not necessarily in sequence) by chance, in tTAV. We also have ubi-rpr.sup.KR constructs of this type (LA3014 and LA3166), which also validates the ubiquitin fusion method described above. The ubiquitin fusion method is further exemplified by RT-PCR analysis of LA3054, LA3056 and LA3488 (FIGS. 11, 13, 14), as described in Example 16, above.

Example 17: Further Cctra Exemplification

(242) Reference is also made to LA3014 and LA3166 and phenotypic data therefrom in other Examples.

(243) We have previously made, and have obtained transgenics with, the Cctra intron in a functional protein other than tTAV, see LA3014 and LA3166. LA3014 contains a ubiquitin-reaper.sup.KR fusion downstream of a Cctra intron. Phenotypic data shows that LA3014 transgenic Medfly gave repressible female-specific lethality. RT-PCR analysis on RNA extracted from adult males and females raised off tetracycline, using primers and ReaperKR, demonstrate that correct splicing was occurring in females (508 bp band) and no such band was found in males (FIG. 37). LA3166 is another construct with the Cctra intron placed inside the ubiquitin coding region fused to reaper.sup.KR, but placed in a different position in ubiquitin. LA3166 also produces a dominant repressible female-specific lethal effect in Medfly.

(244) LA1038 is a new example of the use of the Cctra intron in a different sequence context, here placed in a fragment of Nipp1Dm called nipper that also splices correctly in transgenic Medfly when analysed by RT-PCR (FIG. 12). LA670 was required as a source of tTAV to drive expression of the alternatively spliced nipper.

(245) We have also newly made, and have obtained transgenics with, intron-only Cctra-based constructs with the intron in a different gene (many of the above examples, unless otherwise apparent, are in tTAV or one of its variants, i.e. tTAV2 or tTAV3). These constructs work as predicted. This is an important result, thus showing that there are not essential exonic sequences in Cctra that we have simply duplicated (in function, if not necessarily in sequence) by chance, in tTAV. We also have ubi-rpr.sup.KR constructs of this type (LA3014 and LA3166), which also validates the ubiquitin fusion method described above. The ubiquitin fusion method is further exemplified by RT-PCR analysis of LA3054, LA3056 and LA3488 (FIGS. 11, 13, 14), and as described in Example 16, above.

(246) FIG. 11: Gel showing sex-specific splicing of intron(s) derived from Cctra (780 bp band in females) in Ceratitis capitata transformed with LA3488. Splicing in the F1 form would yield a product of approximately 780 bp. A band of this size is clearly visible from females (lane 4), but not from males, nor in the lanes with reactions from which the reverse transcriptase enzyme was omitted (no RT). Therefore, the Cctra-derived intron is capable of sex-specific alternative splicing in this novel sequence context. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.8, 1.0 and 1.5 kb are indicated); Lanes 2 and 3: Ceratitis capitata LA3488/+ males (RT and no RT control, respectively); Lanes 4 and 5: Ceratitis capitata LA3488/+ females (RT and noRT control, respectively).

(247) FIG. 12: Gel showing sex-specific splicing of intron(s) derived from Cctra in Ceratitis capitata transformed with LA1038. Splicing in the F1 form would yield a product of approximately 230 bp. A band of this size is clearly visible from females (lanes 1, 2, 7, 8, 9 and 10), but not from males. Therefore, the Cctra-derived intron is capable of sex-specific alternative splicing in this novel sequence context. Lane 15: Marker (SmartLadder? from Eurogentec, bands of approx 0.2, 0.4 and 0.6 kb are indicated); Lanes 1, 2, 7, 8, 9 and 10: Ceratitis capitata LA670; LA1038 females; Lanes 3, 4, 5, 6, 11, 12, 13 and 14: Ceratitis capitata LA670; LA1038 males.

(248) FIG. 13: Gel showing sex-specific splicing of intron(s) derived from CcTra in Ceratitis capitata transformed with LA3054. Splicing in the F1 form would yield a product of approximately 340 bp. A band of this size is clearly visible in lane 7, but not from males. Therefore, the Cctra-derived intron is capable of sex-specific alternative splicing in this novel sequence context. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.4, 0.6, 0.8 and 1.0 kb are indicated); Lanes 2-5: Ceratitis capitata LA3054 males; Lane 7: Ceratitis capitata LA3054 female.

(249) FIG. 14: Gel showing sex-specific splicing of intron(s) derived from Cctra in Ceratitis capitata transformed with LA3056. Splicing in the F1 form would yield a product of approximately 200 bp. A band of this size is clearly visible from a female (lane 6), but not from males (lanes 2-4). Therefore, the Cctra-derived intron is capable of sex-specific alternative splicing in this novel sequence context. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.2, 0.4, 0.6 and 0.8 kb are indicated); Lanes 2-5: Ceratitis capitata LA3056/+ males; Lanes 6-7: Ceratitis capitata LA3056/+ females.

(250) FIG. 15: Gel showing sex-specific splicing of intron(s) derived from BzTra in Anastrepha ludens transformed with LA3376. Splicing in the F1 form would yield a product of approximately 672 bp. A band of this size is clearly visible from females (lane 4), but not from males, nor in the lanes with reactions from which the reverse transcriptase enzyme was omitted (no RT), primers used were SRY and AV3F. Therefore, the Bztra-derived intron is capable of sex-specific alternative splicing in this novel sequence context and species. Lane 1: Marker (SmartLadder? from Eurogentec, bands of approx 0.6, 0.8, and 1.0 kb are indicated); Lanes 2 and 3: Anastrepha ludens LA3376/+ males (RT and no RT control, respectively); Lanes 4 and 5: Anastrepha ludens LA3376/+ females (RT and no RT control, respectively).

(251) FIG. 18 and SE ID NOs 149 and 150 show DSX minigene1, DSX minigene2 sequences and LA3619 plasmid map.

(252) FIGS. 19-51 are as per Examples 1-9 above. FIGS. 52-58, 68 and 69 show various plasmid diagrams and sequences. FIGS. 59-60 are described above and FIGS. 61-66 show various further plasmid diagrams and sequences. FIG. 67 is pLA3596, as discussed elsewhere.

REFERENCES

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SEQUENCE ANNOTATIONS

(254) The following relates to the various plasmids of the present and highlights the position of certain preferred elements therein.

(255) <223> Sequence of pLA3359 (SED ID NO. 47).

(256) <***> Key features include:

(257) 1. Anopheles gambiae dsx (Agdsx) mini-gene, [a mini-gene is a recombinant sequence derived from a particular gene (the Agdsx gene in this example) by ligating together non-contiguous segments while retaining original 5-3 order; this is equivalent to deletion of some internal segments from a longer fragment of genomic sequence derived from the gene], (1-3135): including Agdsx part of exon3, exon 4a (female), exon 4b (female) and part of exon5 (male and female).

(258) <***> Exons derived from Agdsx from positions 426 to 560 (part of exon 3); 1068 to 2755 (including part of exon 4, found in females); 1809 to 2755 (including part of exon 4, found in females); and 2914 to 3135 (including part of exon 5, found in males).

(259) <***> Alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?8031 (Ie1 fragment is from position 7431 to 8060).

(260) <***> Included feature:

(261) 1. additional intron derived from Drosophila scraps gene (scraps intron) upstream to Agdsx sequence from position 8075 to 8137.

(262) <223> Sequence of pLA3433 (SED ID NO. 48).

(263) <***> Key features include:

(264) 1. Agdsx mini-gene (778-4623): including Agdsx part of exon 2, exon3, exon 4a (female), exon 4b (female) and part of exon5 (male and female).

(265) <***> Exons derived from Agdsx from position 778 to 908 (part of exon 2); 1913 to 2048 (part of exon 3); 2556 to 2642 (part of exon 4a); 3297 to 4243 (part of exon 4b) and 4402 to 4623 (part of exon 5).

(266) <***> Alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?606 (Ie1 fragment is from position 6 to 635).

(267) <***> Included feature:

(268) 1. additional intron derived from Drosophila scraps gene (scraps intron) upstream to Agdsx sequence from position 650 to 712.

(269) <223> Sequence of pLA3491.

(270) <***> Key features include:

(271) 1. Aedes aegypti dsx (Aadsx) mini-gene: including part of Aadsx exon 4, exon5a (female), exon 5b (female), and part of exon6 (male and female).

(272) <***> Exons derived from Aadsx from position 1316 to 1450 (part of exon 4); 2626 to 3761 (part of exon 5a); 3293 to 3761 (part of exon 5b); and 5215 to 5704 (part of exon 6).

(273) <***> Part of the F1 transcript is predicted to comprise nucleotides ?1174-1450, 2626-3761, 5215-?5850.

(274) <***> Part of the F2 transcript is predicted to comprise nucleotides ?1174-1450, 3293-3761, 5215-?5850.

(275) <***> Part of the F3 transcript is predicted to comprise nucleotides ?1174-1450, 2626-3083, 3293-3761, 5215-?5850.

(276) <***> Part of the M1 transcript is predicted to comprise nucleotides ?1174-1450, 5215-?5850.

(277) <***> Alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?1174 (Ie1 fragment is from position 574 to 1203).

(278) <***> Included feature:

(279) 1. additional intron derived from Drosophila scraps gene (scraps intron) upstream to Aadsx sequence from position 1218 to 1280.

(280) <223> Sequence of pLA3646.

(281) <***> Key features include:

(282) 1. Aadsx mini-gene (17218-11707): including part of Aadsx exon 4 from position 17113 to 16979, exon 5a from position 15803 to 15025+14010 to 13650, exon 5b from position 15136 to 15025+14010 to 13650 and exon 6 from position 12196 to 11707 (note: reverse orientation).

(283) <***> part of exon 4 contains 4 point mutations relative to wild type at positions 17087 (ATG-ACG), 17053 (ATG-ACG), 17050 (ATG-ACG) and 17041 (ATG-ACG) (note: reverse orientation); part of exon 5a and 5b contain 3 point mutations relative to wild type at positions 15129 (ATG-ATA), 15116 (ATG-ATA) and 15113 (ATG-ATA) (note: reverse orientation). All of these mutations are to eliminate ATG sequences.
<***> tTAV2 is inserted in the overlapping exons 5a and 5b from position 15024 to 14011 (note: reverse orientation).
<***> Alternatively spliced transcript starts in hsp70 derived fragment at position ?17312 (hsp70 fragment is from position 17354 to 17225); (note: reverse orientation).
<***> Included feature:

(284) 1. additional intron derived from Drosophila scraps gene (scraps intron) upstream to Aadsx sequence from position 1107 to 1045 (note: reverse orientation)

(285) Sequence of pLA3435 (SED ID NO. 46).

(286) <223> Key features include:

(287) 1. Bombyx mori dsx (Bmdsx) minigene (1411-3161) with an exogenous linker between fused female exons 3 and 4.

(288) <***> Fragment of shared exon two (1411 bp-1554 bp)

(289) <***> Part of female specific exon three (2121 bp-2202) fused to part of female specific exon 4 (2225 bp-2290 bp) using an exogenous linker (2203 bp-2224 bp)

(290) <***> Fragment of shared exon five (3007 bp-3161 bp)

(291) <***> A female dsx mini-gene splicing product is encoded by 1411-1554+2121-2290+3007-3161.

(292) <***> A male dsx mini-gene splicing product is encoded by 1411-1554+3007-3161.

(293) <***> Transcription is predicted to start at approximately position ?1239 within the segment derived from baculovirus AcMNPV Ie1 (immediate early 1) promoter (639 bp-1268 bp).

(294) <223> Sequence of pLA3534.

(295) <***> Key features include:

(296) 1. Aadsx mini-gene (6996-4425): containing Aadsx exon 4, part of exon5a (female) and part of exon 56 (female), inclusive of Aadsx intron fragments.

(297) <***> Exons derived from Aadsx from position 6968 to 6834 (part of exon 4), 5462 to 4425 (part of exon 5a) and 4795 to 4425 (part of exon 5b); (note reverse orientation).

(298) <***> Part of the F1 transcript is predicted to comprise nucleotides ?7146-6834, 5462-4300 (note: reverse orientation).

(299) <***> Part of the F2 transcript is predicted to comprise nucleotides ?7146-6834, 4795-4300 (note: reverse orientation).

(300) <***> Part of the F3 transcript is predicted to comprise nucleotides ?7146-6834, 5462-5005, 4795-4300 (note: reverse orientation).

(301) <***> Alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?7146 (Ie1 fragment is from position 7746 to 7117, reverse orientation).

(302) <223> Sequence of pLA3612.

(303) <***> Key features include:

(304) 1. Ubiquitin-tTAV2 coding region inserted into a female exon of Aadsx gene.

(305) <***> Ubiquitin-tTAV2 is from position 15185-16429 in Aadsx (ubiquitin is from 15185-15412; tTAV2 is from 15413-16429), inclusive of start and stop codon.

(306) <***> Sequence derived from Aadsx: 13150-15184, 16438-18805.

(307) <***> Aadsx-ubiquitin-tTAV2 alternatively spliced transcript starts in hsp70 derived segment (hsp70 fragment is from 13014-13143).

(308) <223> Sequence of pLA3619.

(309) <***> Key features include:

(310) 1. tTAV2 coding region inserted into a female exon of Aadsx gene.

(311) <***> Sequence derived from Aadsx: 5635-3641, 2610-243 (note: reverse orientation).

(312) <***> Aadsx-tTAV2 alternatively spliced transcript starts in hsp70 derived segment from 5642-5771 (note: reverse orientation).

(313) <***> tTAV2 transcript is predicted to be translated between 2619-3635, inclusive of start and stop codon (note: reverse orientation).

(314) <223> Sequence of pLA3545.

(315) <***> Key features include:

(316) 1. AaActin4 promoter and 5 UTR including first intron regulates tTAV expression.

(317) <***> Sequence derived from AaActin4 is from position 923-4285.

(318) <***> Alternatively spliced transcript is predicted to start from approximately ?2366.

(319) <***> The first intron from AaActin4 (female splice variant) is from 2458-4259.

(320) <***> tTAV is predicted to be translated between 4300-5316, inclusive of start and stop codon.

(321) <223> Sequence of pLA3604.

(322) <***> Key features include:

(323) 1. AaActin4 promoter and 5 UTR regulates ubiquitin-tTAV2 expression.

(324) <***> Sequence derived from AaActin4 is from position 5795-2407 (note: reverse orientation).

(325) <***> Alternatively spliced transcript is predicted to start from approximately ?4353 (note: reverse orientation).

(326) <***> The first intron from AaActin4 (female splice variant) is from 2455-4254 (note: reverse orientation).

(327) <***> Ubquitin-tTAV2 transcript is predicted to be translated from a start codon engineered in the first exon of AaAct4 gene at 4299-4297 (ubiquitin is from 2406-2179; tTAV2 is from 2178-1162); (note: reverse orientation).

(328) <223> Sequence of pLA3641.

(329) <***> Key features include:

(330) 1. tTAV coding region inserted into a female exon of CodlingDsx gene.

(331) <***> tTAV is from position 2731-3747 in CodlingDsx gene.

(332) <***> Dsx-tTAV alternatively spliced transcript starts in hsp70 derived segment (hsp70 fragment is from 4811-4940).

(333) <***> tTAV transcript is predicted to be translated between 2731-3747, inclusive of start and stop codon (note: reverse orientation).

(334) <223> Sequence of pLA3570

(335) <***> Key features include:

(336) 1. tTAV coding region inserted into a female exon of PBW-Dsx gene.

(337) <***> tTAV coding region is from 2336-3352.

(338) <***> Dsx-tTAV alternatively spliced transcript starts in hsp70 derived segment (hsp70 fragment is from 4683-4812).

(339) <***> tTAV transcript is predicted to be translated between 2336-3352, inclusive of start and stop codon (note: reverse orientation).

(340) <223> Sequence of pLA1188 (SED ID NO. 49)

(341) <***> Key features include:

(342) 1. tTAV coding region with inserted Cctra intron.

(343) <***> Cctra intron is from position 3905-2561 in tTAV (note: reverse orientation).

(344) <***> tTAV alternatively spliced transcript starts in hsp70 derived segment at position 4217 (hsp70 fragment is from 4260-4131); (note: reverse orientation).

(345) <***> tTAV F1 transcript is predicted to be translated between 4040-1679 (note: reverse orientation).

(346) <***> Included feature:

(347) 1. Adh intron within predicted F1 transcript from position 4118-4049 (note: reverse orientation).

(348) <223> Sequence of pLA3077 (SED ID NO. 50).

(349) <***> Key features include:

(350) 1. tTAV coding region with inserted Cctra intron.

(351) <***> Cctra intron is from position 3975-2631 in tTAV (note: reverse orientation).

(352) <***> tTAV alternatively spliced transcript starts in hsp70 derived segment at position ?4217 (hsp70 fragment is from 4260-4131); (note: reverse orientation).

(353) <***> tTAV F1 transcript is predicted to be translated between 4039-1678, inclusive of start and stop codon (note: reverse orientation).

(354) <***> Included feature:

(355) 1. Adh intron within predicted F1 transcript from position 4117-4048 (note: reverse orientation).

(356) <223> Sequence of pLA3097 (SED ID NO. 51).

(357) <***> Key features include:

(358) 1. tTAV coding region with inserted Cctra intron.

(359) <***> Cctra intron is from position 3282-1938 in tTAV (note: reverse orientation).

(360) <***> tTAV alternatively spliced transcript starts in hsp70 derived segment at position ?3382 (hsp70 fragment is from 3425-3296); (note: reverse orientation).

(361) <***> tTAV F1 transcript is predicted to be translated between 3285-924, inclusive of start and stop codon (note: reverse orientation).

(362) <223> Sequence of pLA3233 (SED ID NO. 52).

(363) <***> Key features include:

(364) 1. tTAV2 coding region with inserted Cctra intron.

(365) <***> Cctra intron is from position 3289-1945 in tTAV2 (note: reverse orientation).

(366) <***> tTAV2 alternatively spliced transcript starts in hsp70 derived segment at position ?3389 (hsp70 fragment is from 3432-3303); (note: reverse orientation).

(367) <***> tTAV2 F1 transcript is predicted to be translated between 3292-931, inclusive of start and stop codon (note: reverse orientation).

(368) <223> Sequence of pLA3014 (SED ID NO. 53).

(369) <***> Key features include:

(370) 1. ubi-reaper[KR] coding region with inserted Cctra intron.

(371) <***> Cctra intron is from position 3356-4700 in ubi-reaper[KR].

(372) <***> ubi-reaper[KR] alternatively spliced transcript starts in hsp70 derived segment at position ?3234 (hsp70 fragment is from 3191-3320).

(373) <***> ubi-reaper[KR] F1 transcript is predicted to be translated between 3331-5143, inclusive of start and stop codon (ubiquitin is from 3331-3355, 4701-4948; reaper[KR] is from 4949-5143).

(374) <223> Sequence of pLA3166 (SED ID NO. 54).

(375) <***> Key features include:

(376) 1. ubi-reaper[KR] coding region with inserted Cctra intron.

(377) <***> Cctra intron is from position 9987-8643 in ubi-reaper[KR] (note: reverse orientation).

(378) <***> ubi-reaper[KR] alternatively spliced transcript starts in hsp70 derived segment at position ?10227 (hsp70 fragment is from 10270-10141); (note: reverse orientation).

(379) <***> ubi-reaper[KR] F1 transcript is predicted to be translated between 10126-8359, inclusive of start and stop codon (ubiquitin is from 10126-9988, 8642-8554; reaper[KR] is from 8553-8359); (note: reverse orientation).

(380) <223> Sequence of pLA3376 (SED ID NO. 55).

(381) <***> Key features include:

(382) 1. tTAV2 coding region with inserted Cctra intron.

(383) 2. tTAV3 coding region with inserted Bztra intron.

(384) 3. reaper[KR] coding region with inserted Bztra intron.

(385) <***> Cctra intron is from position 3289-1945 in tTAV2 (note: reverse orientation).

(386) <***> Bztra intron is from position 5981-5014 in tTAV3 (note: reverse orientation).

(387) <***> Bztra intron is from position 16391-17358 in reaper[KR].

(388) <***> tTAV2 alternatively spliced transcript starts in hsp70 derived segment at position ?3389 (hsp70 fragment is from 3432-3303); (note: reverse orientation).

(389) <***> tTAV3 alternatively spliced transcript starts in sry-alpha derived segment at position ?6019 (sry-alpha fragment is from 6243-5999); (note: reverse orientation).

(390) <***> reaper[KR] alternatively spliced transcript starts in hunchback derived segment at position ?16339 (hunchback fragment is from 16289-16372).

(391) <***> tTAV2 F1 transcript is predicted to be translated between 3292-931, inclusive of start and stop codon (note: reverse orientation).

(392) <***> tTAV3 F1 transcript is predicted to be translated between 5984-4006, inclusive of start and stop codon (note: reverse orientation).

(393) <***> reaper[KR] F1 transcript is predicted to be translated between 16385-17550, inclusive of start and stop codon.

(394) <223> Sequence of pLA3242 (SED ID NO. 56).

(395) <***> Key features include:

(396) 1) tTAV coding region with inserted Cctra intron.

(397) 2) reaper[KR] coding region with inserted Crtra intron.

(398) <***> Cctra intron is from position 3282-1938 in tTAV (note: reverse orientation).

(399) <***> Crtra intron is from position 5488-4180 in reaperKR (note: reverse orientation).

(400) <***> reaperKR alternatively spliced transcript starts in hunchback derived segment at position ?5540 (hunchback fragment is from 5590-5507); (note: reverse orientation).

(401) <***> tTAV alternatively spliced transcript starts in hsp70 derived segment at position ?3382 (hsp70 fragment is from 3425-3296); (note: reverse orientation).

(402) <***> reaperKR F1 transcript is predicted to be mainly translated between 4088-5494, inclusive of start and stop codon (note: reverse orientation).

(403) <***> tTAV F1 transcript is predicted to be mainly translated between 924-3285, inclusive of start and stop codon (note: reverse orientation).

(404) <223> Sequence of pLA1172 (SED ID NO. 106).

(405) <***> Key features include:

(406) 1. tTAV coding region between AaActin4 derived fragments.

(407) <***> AaActin4 derived fragments are from 7868-11257 and 12366-13100.

(408) <***> tTAV transcript is predicted to be translated between 11342-12358, inclusive of start and stop codon.

(409) <***> AaActin4-tTAV transcript is predicted to start at position ?9312.

(410) <***> AaActin4 contains an intron (female-type splice variant) from position 9403-11204.

(411) <223> Sequence of pLA1038 (FIG. 12).

(412) <***> Key features include:

(413) 1. Fragment of Nipp1Dm (nipper) coding region with inserted Cctra intron with flanking tra exonic sequence.

(414) <***> Cctra intron is from position 3365-4709 in nipper.

(415) <***> Cctra intron is flanked by Cctra exonic sequence at positions 3343-3364 and 4710-4729.

(416) <***> nipper alternatively spliced transcript starts in hsp70 derived segment at position ?3243 (hsp70 fragment is from 3200-3329).

(417) <***> nipper F1 transcript is predicted to be translated between 3340-5014, inclusive of start and stop codon.

(418) <223> Sequence of pLA3054 (SED ID NO. 158).

(419) <***> Key features include:

(420) 1. DsRed-ubi-tTAV coding region with inserted Cctra intron with flanking tra exonic sequence.

(421) <***> Cctra intron is from position 3509-2165 in DsRed-ubi-tTAV (note: reverse orientation).

(422) <***> Cctra intron is flanked by Cctra exonic sequence at positions 3531-3510 and 2164-2145 (note: reverse orientation).

(423) <***> DsRed-ubi-tTAV alternatively spliced transcript starts either in hsp70 derived segment at position ?3243 (hsp70 fragment is from 4930-4801) or Opie2 derived segment at position ?4353 (Opie2 fragment is from 4795-4255); (note: reverse orientation).

(424) <***> DsRed-ubi-tTAV F1 transcript is predicted to be translated between 4320-888, inclusive of start and stop codon (DsRed is from 4212-3538; ubiquitin is from 2135-1908; tTAV is from 1907-888); (note: reverse orientation).

(425) <223> Sequence of pLA3056 (SED ID NO. 159).

(426) <***> Key features include:

(427) 1. DsRed-ubi-tTAV coding region with inserted Cctra intron with flanking tra exonic sequence.

(428) <***> Cctra intron is from position 3731-2387 in DsRed-ubi-tTAV (note: reverse orientation).

(429) <***> Cctra intron is flanked by Cctra exonic sequence at positions 3753-3732 and 2386-2145 (note: reverse orientation).

(430) <***> DsRed-ubi-tTAV alternatively spliced transcript starts either in hsp70 derived segment at position ?5109 (hsp70 fragment is from 5152-5023) or Opie2 derived segment at position ?4575 (Opie2 fragment is from 5017-4477); (note: reverse orientation).

(431) <***> DsRed-ubi-tTAV F1 transcript is predicted to be translated between 4542-888, inclusive of start and stop codon (DsRed is from 4434-3760; ubiquitin is from 2135-1908; tTAV is from 1907-888); (note: reverse orientation).

(432) <***> Included feature:

(433) 1. additional intron derived from Cctra gene (second intron of Cctra F1 transcript) within predicted F1 transcript from position 2222-2168 (note: reverse orientation).

(434) <223> Sequence of pLA3488 (SED ID NO. 160).

(435) <***> Key features include:

(436) 1. TurboGreen-ubi-DsRed coding region with inserted Cctra intron.

(437) <***> Cctra intron is from position 2263-3607 in TurboGreen-ubi-DsRed.

(438) <***> TurboGreen-ubi-DsRed alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?1180 (Ie1 fragment is from 580-1209).

(439) <***> TurboGreen-ubi-DsRed F1 transcript is predicted to be translated between 1311-4467, inclusive of start and stop codon (TurboGreen is from 1311-2093; SG4 linker is from 2094-2123; ubiquitin is from 2124-3696, inclusive of Cctra intron; DsRed is from 3697-4467).
<***> Included feature:

(440) 1. additional intron derived from Drosophila scraps gene (scraps intron) within predicted F1 transcript from position 1224-1286.

(441) <223> Sequence of pLA3596 (SED ID NO. 145).

(442) <***> Key features include:

(443) 1. TurboGreen-ubi-DsRed2 coding region with inserted Cctra intron.

(444) <***> Cctra intron is from position 5947-7291 in TurboGreen-ubi-DsRed2.

(445) <***> TurboGreen-ubi-DsRed2 alternatively spliced transcript starts in segment derived from baculovirus AcMNPV Ie1 (immediate early 1) at position ?4864 (Ie1 fragment is from 4264-4893).

(446) <***> TurboGreen-ubi-DsRed2 F1 transcript is predicted to be translated between 4995-8148, inclusive of start and stop codon (TurboGreen is from 4995-5777; SG4 linker is from 5778-5807; ubiquitin is from 5808-7380, inclusive of Cctra intron; DsRed2 is from 7381-8151).
<***> Included feature:

(447) 1. additional intron derived from Drosophila scraps gene (scraps intron) within predicted F1 transcript from position 4908-4970.

(448) 2. intended amino acid mutation compared to LA3488 at position 7294-7296.