METHOD FOR THE GENOTYPING OF MOUSE STRAINS
20230183781 · 2023-06-15
Assignee
Inventors
Cpc classification
C12Q1/6888
CHEMISTRY; METALLURGY
C12Q1/6876
CHEMISTRY; METALLURGY
International classification
Abstract
The invention relates in general to the analysis of genetic markers in individuals of non-human origin, in particular the genotyping of outbred populations of rodents or fish. The invention relates specifically to the simultaneous amplification of at least five different polymorphic autosomal markers and at least one polymorphic Y-chromosomal marker of the mouse in a reaction preparation by means of polymerase chain reaction or other multiplex methods and the detection of the specific alleles for each marker of the multiplex method. The invention further relates to a kit for genetically identifying and/or for differentiating two or more animals from DNA extracts of individuals from wild-type populations, from different inbred or outbred strains, of an identical inbred or outbred strain, or from substrains of an identical inbred or outbred strain, in particular of the rat or mouse.
Claims
1. A method for genetically identifying and/or for distinguishing two or more animals of the species mouse, rat, hamster and zebrafish from different outbred strains, of the same outbred strain or of substrains of the same outbred strain, comprising the following substeps: (a) using at least one DNA sample which is to be analyzed; (b) selecting a set of loci for the DNA sample which comprises at least 5, preferably at least 6, 7 or 8, particularly preferably at least 9 or 10, most preferably at least 11 autosomal STR markers which are coamplified simultaneously in a reaction mix; (c) selecting a set of loci for the DNA sample which comprises at least two Y-chromosomal STR markers which are coamplified simultaneously with the autosomal STR loci (b) in a reaction mix; (d) using a set of oligonucleotide primer pairs in which each primer pair binds specifically to the flanking DNA segments of an STR marker from the sets of STR loci (b) and (c); (e) coamplifying the loci selected for the set, the reaction product being a mixture consisting of amplified alleles of each of the coamplified STR loci of the sets (b) and (c); (f) analyzing the allele mixture with the goal of determining and assigning the specific alleles for each of the amplified loci of the sets (b) and (c) for the given DNA sample; the autosomal STR loci being characterized by a high value for the “polymorphism information content”, which is within the range of 0.30 to 0.99, preferably within the range of 0.50 to 0.99, particularly preferably within the range between 0.60 to 0.99.
2. The method as claimed in claim 1, characterized in that the DNA sample originates from at least one animal of the species mouse, rat, hamster or zebrafish or from a cell line of the species mouse, rat, hamster or zebrafish.
3. The method as claimed in claim 1, characterized in that at least two of the eleven autosomal STR loci (b) are selected from the group comprising the loci D1Mmu121 (SEQ ID NO: 51), D2Mmu008 (SEQ ID NO: 52), D3Mmu158 (SEQ ID NO: 53), D4Mmu155 (SEQ ID NO: 54), D5Mmu108 (SEQ ID NO: 55), D6Mmu120 (SEQ ID NO: 56), D7Mmu003 (SEQ ID NO: 57), D8Mmu127 (SEQ ID NO: 58), D9Mmu100 (SEQ ID NO: 59), D10Mmu043 (SEQ ID NO: 60), D11Mmu030 (SEQ ID NO: 61), D12Mmu056 (SEQ ID NO: 62), D13Mmu096 (SEQ ID NO: 63), D14Mmu074 (SEQ ID NO: 64), D15Mmu084 (SEQ ID NO: 65), D16Mmu030 (SEQ ID NO: 66), D17Mmu041 (SEQ ID NO: 67), D18Mmu069 (SEQ ID NO: 68) and D19Mmu008 (SEQ ID NO: 69).
4. The method as claimed in claims 1 and 3, characterized in that the set of eleven autosomal loci in step (b) comprises the loci D2Mmu008 (SEQ ID NO: 52), D3Mmu158 (SEQ ID NO: 53), D4Mmu155 (SEQ ID NO: 54), D6Mmu120 (SEQ ID NO: 56), D7Mmu003 (SEQ ID NO: 57), D8Mmu127 (SEQ ID NO: 58), D10Mmu043 (SEQ ID NO: 60), D13Mmu096 (SEQ ID NO: 63), D14Mmu074 (SEQ ID NO: 64), D16Mmu030 (SEQ ID NO: 66) and D18Mmu069 (SEQ ID NO: 68).
5. The method as claimed in claim 1, further comprising sex identification in the DNA sample of the individual in step (a) on the basis of the set of loci according to step (c).
6. The method as claimed in claim 5, further comprising distinguishing between Y chromosomes of the type Mus musculus musculus and of the type Mus musculus domesticus on the basis of the set of loci according to step (c).
7. The method as claimed in claim 5, characterized in that the locus for sex determination is a polymorphic STR locus of mouse that can distinguish Y-chromosomal haplotypes and is selected from a group of loci listed as follows: DYMmu001 (SEQ ID NO: 70), DYMmu002 (SEQ ID NO: 71) and DYMmu003 (SEQ ID NO: 72).
8. The method as claimed in claim 5, characterized in that the locus for sex determination is a polymorphic STR locus of rat that can distinguish Y-chromosomal haplotypes and is selected from a group of loci listed as follows: DYRno004 (SEQ ID NO: 73 and SEQ ID NO: 74), DYRno165 (SEQ ID NO: 75) and DYRno304 (SEQ ID NO: 76).
9. The method as claimed in claim 1, characterized in that a multiplex amplification with at least thirteen pairs of oligonucleotide primers, which comprises at least eleven autosomal and two Y-chromosomal STR loci, is carried out.
10. The method as claimed in claim 1, characterized in that the amplified alleles are separated from one another beforehand by means of an analytical or semipreparative method before the evaluation in step (f).
11. The method as claimed in claim 10, characterized in that at least one oligonucleotide primer of a primer pair is covalently coupled to a detection dye, preferably to a fluorescent dye.
12. The method as claimed in claim 11, characterized in that at least four different dye-coupled primers with four different detection dyes, preferably four different fluorescent dyes, are used.
13. The method as claimed in claim 1, characterized in that the assignment of the amplified alleles in step (f) is done on the basis of comparison with a size standard, the size standard being a mixture of DNA fragments of known size and/or a locus-specific mixture of known alleles.
14. A kit for the genetic identification and/or for the distinguishing of two or more animals from DNA extracts of individuals from wild-type populations, from different inbred or outbred strains, of the same inbred or outbred strain or of substrains of the same inbred or outbred strain of mouse, comprising: (a) a set of oligonucleotide primer pairs in which each primer pair binds specifically to the flanking DNA segments of a locus from the set of STR loci and which comprises at least 5, preferably at least 6, 7 or 8, particularly preferably at least 9 or 10, most preferably at least 11 autosomal STR markers which can be coamplified simultaneously in a reaction mix; (b) a set of oligonucleotide primer pairs which bind specifically to the flanking DNA segments of the Y chromosome and which comprises at least two, preferably three Y-chromosomal STR markers which can be coamplified simultaneously with the autosomal STR loci (a) in a reaction mix; (c) reagents which are sufficient for carrying out at least one multiplex PCR; and (d) a size standard which contains different DNA markers of a known fragment length.
15. The kit as claimed in claim 14, further comprising: (e) a size standard which contains a locus-specific mixture of known alleles.
16. The kit as claimed in claim 14, characterized in that at least two of the eleven autosomal STR loci (a) are selected from the group comprising the loci D1Mmu121 (SEQ ID NO: 51), D2Mmu008 (SEQ ID NO: 52), D3Mmu158 (SEQ ID NO: 53), D4Mmu155 (SEQ ID NO: 54), D5Mmu108 (SEQ ID NO: 55), D6Mmu120 (SEQ ID NO: 56), D7Mmu003 (SEQ ID NO: 57), D8Mmu127 (SEQ ID NO: 58), D9Mmu100 (SEQ ID NO: 59), D10Mmu043 (SEQ ID NO: 60), D11Mmu030 (SEQ ID NO: 61), D12Mmu056 (SEQ ID NO: 62), D13Mmu096 (SEQ ID NO: 63), D14Mmu074 (SEQ ID NO: 64), D15Mmu084 (SEQ ID NO: 65), D16Mmu030 (SEQ ID NO: 66), D17Mmu041 (SEQ ID NO: 67), D18Mmu069 (SEQ ID NO: 68) and D19Mmu008 (SEQ ID NO: 69).
17. The kit as claimed in claim 14, characterized in that the set of eleven autosomal loci in step (a) comprises the loci D2Mmu008 (SEQ ID NO: 52), D3Mmu158 (SEQ ID NO: 53), D4Mmu155 (SEQ ID NO: 54), D6Mmu120 (SEQ ID NO: 56), D7Mmu003 (SEQ ID NO: 57), D8Mmu127 (SEQ ID NO: 58), D10Mmu043 (SEQ ID NO: 60), D13Mmu096 (SEQ ID NO: 63), D14Mmu074 (SEQ ID NO: 64), D16Mmu030 (SEQ ID NO: 66) and D18Mmu069 (SEQ ID NO: 68).
18. The kit as claimed in any of claim 14, characterized in that the set of Y-chromosomal loci (b) comprises the loci DYMmu001 (SEQ ID NO: 70), DYMmu002 (SEQ ID NO: 71) and DYMmu003 (SEQ ID NO: 72).
Description
[0089] What is shown by:
[0090]
EXEMPLARY EMBODIMENT 1: ASSIGNMENT OF THE Y CHROMOSOMES IN SELECTED OUTBRED POPULATIONS TO THE TYPES M. M. MUSCULUS AND M. M. DOMESTICUS
[0091] Describing the genetic diversity of an outbred population requires knowledge of the number of different variants of the Y chromosome in the population. According to the invention, this object is achieved by first performing an assignment of the Y chromosomes of outbred populations to the two types M. m. domesticus and M. m. musculus. This requires markers which can assign a Y chromosome to one of the two basic variants. Surprisingly, it was possible to identify the STR marker DYMmu003, which has different alleles in different variants of M. m. musculus. By contrast, the PCR primers used do not have a homologous sequence for all variants of the Y chromosome of the type M. m. domesticus. The result of this is that, in the case of the presence of a chromosome of the type M. m. domesticus, no PCR product is generated. DYMmu003 is utilized to establish the belonging of Y chromosomes to one of the two types. To this end, between 5 and 25 male individuals of the outbred populations Hsd:ICR(CD1), Crl:CD1(ICR), RjOrl:SWISS, Crl:NMRI, Han:NMRI, RjHan:NMRI and HsdWin:NMRI were analyzed in each case. Altogether 100 male animals of 7 different outbred populations of mouse were analyzed using the STR marker DYMmu003 and assigned accordingly to M. m. musculus or M. m. domesticus (Table 3). In the case of type M. m. domesticus, DNA products are not obtained for either of the two markers; in the case of type M. m. musculus, alleles are always detected. Completely unexpectedly, only the outbred population Crl:NMRI has Y chromosomes of both types, whereas the outbred populations Hsd:ICR(CD1), Crl:CD1(ICR), RjOrl:SWISS and RjHan:NMRI have solely variants of the type M. m. domesticus and Han:NMRI and HsdWin:NMRI can always be assigned only to the type M. m. musculus.
TABLE-US-00003 TABLE 3 Assignment of Y chromosomes to the type M. m. domesticus and M. m. musculus in various outbred populations (N = number of tested individuals) M. m. M. m. N domesticus musculus Hsd:ICR(CD1) 25 25 0 Crl:CD1(ICR) 5 5 0 RjOrl:SWISS 20 20 0 Crl:NMRI 20 17 3 Han:NMRI 20 0 20 RjHan:NMRI 5 5 0 HsdWin:NMRI 5 0 5
EXEMPLARY EMBODIMENT 2: ASSIGNMENT OF ALLELES OF THE STR MARKERS DYMMU001, DYMMU002 AND DYMMU003 OF THE Y CHROMOSOME IN SELECTED OUTBRED POPULATIONS
[0092] It is further an object of the present invention to make available STR markers for the Y chromosome that are polymorphic both in the case of the type M. m. musculus and in the case of the type M. m. domesticus and can be used for describing Y-chromosomal haplotypes. The inventive STR markers DYMmu001 and DYMmu002 have this property. On the basis of DNA samples from male animals of various inbred strains and outbred populations of mouse, the possible allele spectrum for the markers DYMmu001, DYMmu002 and DYM003 was determined.
[0093] Altogether 50 male animals of 15 different inbred strains and 85 male animals of 7 different outbred populations of mouse were analyzed using the markers DYMmu001, DYMmu002 and DYMmu003 (Table 4). The assignment to M. m. musculus or M. m. domesticus in the case of the outbred populations was done on the basis of the relevant investigation results for DYMmu003. Some alleles were detected solely in inbred strains.
TABLE-US-00004 TABLE 4 Detected alleles of the STR markers DYMmu001, DYMmu002 and DYMmu003 and the assignment thereof in outbred populations to M. m. musculus and M. m. domesticus Outbred Inbred + outbred M. m. M. m. Alleles found domesticus musculus DYMmu001 16, 17, 18, 19, 21, 16, 17, 18, 20, 21, 22, 23 20, 22, 23, 24, 25, 26 21 DYMmu002 17, 19, 20, 21, 23, 17, 23, 24, 25 19, 20 24, 25 DYMmu003 12, 14 No PCR product 14
EXEMPLARY EMBODIMENT 3: DETECTION OF DIFFERENT Y-CHROMOSOMAL HAPLOTYPES WITHIN OUTBRED POPULATIONS OF MOUSE
[0094] Since the Y chromosome does not recombine, the analysis results of different Y-STR markers can be combined with one another and presented in the form of haplotypes. Table 5 presents different Y-chromosomal haplotypes for 7 different outbred populations of CD1, SWISS and NMRI, which were obtained on the basis of the analysis results for the STR markers DYMmu001, DYMmu002 and DYMmu003. With the exception of the outbred population HsdWin:NMRI with 5 identical Y-STR haplotypes among 5 tested individuals, all the other outbred populations have more than one haplotype. According to the invention, it is possible to perform, within a specific outbred population, an assignment of male individuals to different haplotypes and also the assignment to one of the two basic Y-chromosome types M. m. musculus or M. m. domesticus. This approach to describing the genetic diversity of outbred populations via the Y chromosome is novel and can be used in future for genetic monitoring.
[0095] Altogether 100 male animals of 7 different outbred populations of mouse were analyzed using the STR markers DYMmu001, DYMmu002 and DYMmu003. The haplotypes were derived from the results of the analysis of the single samples, the haplotype results being presented in the order DYMmu001-DYMmu002-DYMmu003. The value “0” for DYMmu003 indicates that no PCR product is detectable and that a chromosome of the type M. m. domesticus is concerned. According to the invention, it is possible to detect different combinations of alleles within an outbred population, and this demonstrates the presence of various Y chromosomes within an outbred population that are distinguishable from one another.
[0096] The comparison of haplotypes between various outbred populations shows that there are in some cases haplotypes which are unique for a specific outbred population and were not able to be detected in another outbred population.
TABLE-US-00005 TABLE 5 Y-chromosomal haplotypes in various outbred populations. The order of haplotype indication is DYMmu001-DYMmu002-DYMmu003 Hsd:ICR Crl:CD1 (CD1) (ICR) RjOrl:SWISS Crl:NMRI Han:NMRI RjHan:NMRI HsdWin:NMRI N = 25 N = 5 N = 20 N = 20 N = 20 N = 5 N = 5 16-23-0 17-24-0 16-23-0 19-17-0 21-19-14 17-24-0 23-20-14 16-24-0 17-25-0 17-23-0 20-17-0 21-20-14 17-25-0 17-23-0 18-24-0 17-24-0 21-17-0 17-24-0 22-20-14 17-25-0
EXEMPLARY EMBODIMENT 4: CHARACTERIZATION OF THE RAT Y CHROMOSOME WITH THE AID OF STR MARKERS
[0097] Male animals of 17 different strains of Rattus norwegicus were analyzed using the inventive Y-chromosomal markers DYRno004, DYRno165 and DYRno304. The alleles were designated on the basis of the measured lengths of the amplification products. This is sufficient for documenting the diversity of different alleles. Exact knowledge of the DNA sequences of the individual alleles is not absolutely necessary for this purpose. The haplotypes derived from the analysis results are presented in Table 7 in the order DYRno004, DYRno165 and DYRno304. All the strains are distinguishable from one another. These markers are thus suitable for genotyping the rat Y chromosome and for establishing the genetic diversity within outbred populations of rat. Table 6 brings together the inventive STR markers, the oligonucleotide primers used, the chromosomal location thereof and the structure of the repeat units. For each primer, what is specified is the chromosomal location thereof in base pairs, which was ascertained on the basis of reference DNA (NCBI, version Rnor_6.0). Since the rat Y chromosome is only very short, a detailed assignment was performed to characterize the chromosomal position of the marker. DYRno165 corresponds to the position at 1.65 megabases.
[0098] The oligonucleotide primer pair of the marker DYRno004 binds specifically to two different target regions of the Y chromosome and an allele product is obtained for each target region. The two alleles amplified in this connection usually differ from one another with respect to the number of repeat units, meaning that two different alleles can be detected simultaneously using one primer pair. Assigning the individual alleles to the specific target region is, nevertheless, not possible. The STR marker DYRno004 is thus a double marker which is highly polymorphic and is ideally suitable for creating a Y-specific haplotype. The smaller of the two alleles is listed first in the haplotype, then the second, larger allele. For the two markers DYRno165 and DYRno304, the same reverse primer is used; owing to the different forward primer, it is possible to perform an unambiguous assignment of alleles to the specific marker. According to the invention, the two specific forward primers can be provided with different fluorescent dyes, and this allows an unambiguous assignment of the amplification products.
TABLE-US-00006 TABLE 6 List of the inventive Y-chromosomal STR markers in the case of rat Rattus norwegicus, the oligonucleotide primers used, the location thereof on the Y chromosome in base pairs and the pattern of the STR repeat unit. The number of repeat units is based on the reference sequence deposited in the NCBI database (version Rnor_6.0). The alleles were named according to the guidelines (DNA Recommendations, 1997). (F-forward primer, R-reverse primer) Y-chromosomal position Marker Primer sequence (5′-3′) (bp) Repeat unit DYRno004 SEQ ID NO: F GTCAGAAGGGGCAGACT 49153-49172 CTTT(CCTT)3 45 CTA 249767- (CTTT)18 249748 (see SEQ ID NO: 73 SEQ ID NO: R TCACTTGGGATGGTGAA 49393-49374 and 74) 46 CTG 249523- 249542 DYRno165 SEQ ID NO: F CAGACCTCAGGGCATTT 1657537- (ATCTATGT)7ATCT 47 CCAT 1657557 (TCTA)15 SEQ ID NO: R ATATGGCTCTTTGTTCTC 1657766- (see SEQ ID NO: 75) 48 TGG 1657746 DYRno304 SEQ ID NO: F CAGATCTCAGGGCATTC 3047184- (ATCT) 16 49 CCA 3047203 (see SEQ ID NO: 76) SEQ ID NO: R ATATGGCTCTTTGTTCTC 3047359- 50 TGG 3047339
[0099] Table 7 brings together the analysis results of the genotyping of 17 different strains of Rattus norwegicus using the STR markers DYRno004, DYRno165 and DYRno304. The designation of the alleles corresponds to the measured lengths of the amplification products. In principle, this is sufficient for demonstrating the diversity of different alleles without specific knowledge of the underlying DNA sequence. The presented haplotypes are presented in the order of the analysis results for DYRno004, DYRno165 and DYRno304. Each strain has its own haplotype. The combination of the three inventive STR markers is suitable for representing the genetic diversity of the Y chromosome in the case of rat.
TABLE-US-00007 TABLE 7 Y-chromosomal haplotypes in various strains of rat STR loci Rat strain DYRno004 DYRno165 DYRno304 Haplotype ACI 255-258 243 188 255-258-243-188 BN 254-264 231 184 254-264-231-184 BS 259-264 243 192 259-264-243-192 DA 234-247 247 192 234-247-247-192 E3 259-267 247 192 259-267-247-196 F344 255-267 243 196 255-267-243-196 LE 259-267 247 196 259-267-247-196 LEW 259-264 251 188 259-264-251-188 LOU-C 264-267 247 192 264-267-247-192 MNS 255-271 247 192 255-271-247-192 NAR 255-259 247 192 255-259-247-192 OM 251-254 231 184 251-267-231-184 PAR 251-267 243 196 251-267-243-196 PVG 251-264 251 211 251-264-251-211 WC 264-275 239 196 264-275-239-196 WF 255-255 247 192 255-255-247-192 WKY 259-283 255 188 259-283-255-188
EXEMPLARY EMBODIMENT 5: DETERMINATION OF THE GENOTYPE FOR ANIMALS OF THE OUTBRED POPULATIONS HSD:ICR(CD1) AND RJORL:SWISS
[0100] The genotypes were determined for 20 male animals in each case of the outbred populations Hsd:ICR(CD1) and RjOrl:SWISS, and the PIC and Het values were calculated on the basis thereof. It is essential for the assessment of the quality of a marker that multiple different alleles are detectable and that none of the alleles should represent more than 50% of the alleles in a population. To be able to estimate this, the analysis of 20 animals per population was deemed sufficient. In the case of such a number of animals, there are results across altogether 40 alleles (2 per animal); an allele with a proportion of 10% in the population is, in this connection, detected four times from a statistical point of view.
[0101] The number of possible reference alleles as ascertained following the genotyping of a multiplicity of inbred strains and outbred populations varies, depending on the marker, between 7 and 11. For Hsd:ICR(CD1) and RjOrl:SWISS, STR markers were identified in which it was possible to detect the presence of at least 6 to 9 different alleles among the 20 genotyped animals. The PIC values vary between 0.34 and 0.84. The heterozygosity rate is between 0.50 and 0.95.
TABLE-US-00008 TABLE 8 Characterization of STR markers in RjOrl:SWISS and Hsd:ICR(CD1) on the basis of the number of detected alleles per marker (No.) and the biostatistical parameters “polymorphism information content” (PIC) and heterozygosity (Het). The column “Refer. allele” presents the total number of alleles which were observed altogether in 150 individuals of various inbred and outbred strains and substrains of inbred strains of mouse. The column “No.” indicates how many different alleles were detected among the 20 DNA samples of male individuals of the specific outbred population. Refer. RjOrl:SWISS Hsd:ICR(CD1) STR locus allele No. PIC Het No. PIC Het D1Mmu121 10 8 0.64 0.75 6 0.70 0.85 D2Mmu008 7 6 0.67 0.65 6 0.75 0.75 D3Mmu158 8 7 0.79 0.90 6 0.69 0.60 D4Mmu155 8 6 0.72 80.0 6 0.69 0.75 D5Mmu108 8 6 0.63 70.0 6 0.71 0.95 D6Mmu120 7 7 0.75 0.85 7 0.79 0.80 D7Mmu003 9 6 0.72 0.70 7 0.75 0.85 D8Mmu127 10 7 0.74 0.75 7 0.63 0.65 D9Mmu100 10 5 0.50 0.45 8 0.83 0.90 D10Mmu043 7 7 0.80 0.75 6 0.73 0.70 D11Mmu030 11 6 0.71 0.85 4 0.57 0.75 D12Mmu056 8 7 0.78 0.80 4 0.60 0.70 D13Mmu096 11 9 0.84 0.90 7 0.79 0.85 D14Mmu074 8 6 0.72 0.85 4 0.64 0.75 D15Mmu084 9 9 0.73 0.65 5 0.52 0.50 D16Mmu030 10 6 0.72 0.85 5 0.71 0.75 D17Mmu041 7 5 0.61 0.75 3 0.34 0.55 D18Mmu069 11 7 0.78 0.65 7 0.67 0.70 D19Mmu008 8 7 0.72 0.70 5 0.51 0.65
EXEMPLARY EMBODIMENT 6: ELECTROPHEROGRAM OF A MULTIPLEX PCR OF 14 STR LOCI
[0102]
[0103] The PCR was carried out in a total volume of 25 μl using Snooplex® FastPrep PCR reagents (GVG Genetic Monitoring, Leipzig). The reaction mix for the PCR analysis consisted of the following components: 2.1 μl of nuclease-free water, 5 μl of 5×PCR buffer, 0.8 μl of Taq DNA polymerase, primer mixtures of the STR markers D2Mmu008 (0.8 μl), D3Mmu158 (1.2 μl), D4Mmu155 (0.8 μl), D6Mmu120 (1.6 μl), D7Mmu003 (1.2 μl), D8Mmu127 (1.2 μl), D10Mmu043 (0.4 μl), D13Mmu096 (0.7 μl), D14Mmu074 (1.6 μl), D16Mmu030 (1.6 μl), D18Mmu069 (1.2 μl), DYMmu001 (1.0 μl), DYMmu002 (0.8 μl), DYMmu003 (1.0 μl) and 2 μl of DNA.
[0104] The inventive multiplex PCR shown in exemplary embodiment 6 does not lead to any detectable alleles when using DNA of human origin or of rat, hamster or zebrafish.
REFERENCES
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