Transgenic Insect and Use of Same in Methods for Testing Natural or Synthetic Substances

Abstract

A transgenic insect includes a genome which has at least one first exogenic DNA sequence, which is coded for a human membrane transporter protein. The expression of the first exogenic DNA sequence leads to a functional human membrane transporter protein in the transgenic insect.

Claims

1. A transgenic insect, comprising: a genome including at least one first exogenous DNA sequence encoding a human membrane transporter protein, the expression of the first exogenous DNA sequence leading to a functional human membrane transporter protein in the transgenic insect.

2. The transgenic insect as claimed in claim 1, wherein the expression of the first exogenous DNA sequence of the human membrane transporter protein in the insect is one or more of salivary gland tissue-specific, intestinal system tissue-specific, tracheal tissue-specific, and Malpighian tissue-specific.

3. The transgenic insect as claimed in claim 1, wherein the transgenic insect is a fruit fly belonging to the genus Drosophila.

4. The transgenic insect as claimed in claim 1, wherein the human membrane transporter protein is a human uptake transporter protein or a human efflux transporter protein.

5. The transgenic insect as claimed in claim 1, wherein the expression of the first exogenous DNA sequence encoding a human membrane transporter protein is under the control of one or more of a salivary gland tissue-specific GAL4/UAS, an intestinal system tissue-specific GAL4/UAS, a tracheal tissue-specific GAL4/UAS, and Malpighian tissue-specific GAL4/UAS expression system.

6. The transgenic insect as claimed in claim 1, wherein the genome thereof comprises at least one second exogenous DNA sequence encoding including at least one fluorescent protein selected from the group consisting of GFP, CFP, YFP, mCherry, dsRed, and variants thereof.

7. The transgenic insect as claimed in claim 1, wherein the transgenic insect is a transgenic Drosophila, the genome further including: a vector wherein the human membrane transporter protein is an uptake transporter protein or an efflux transporter protein selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2, and genetic variants thereof.

8. A method for generating a transgenic insect, comprising: subcloning a first exogenous DNA sequence encoding a human membrane transporter protein into an expression vector to obtain a vector comprising the first exogenous DNA sequence; introducing the vector comprising the first exogenous DNA sequence into an insect to obtain a stable strain of a transgenic precursor insect; and crossing the transgenic precursor insect with an insect comprising an expression system matched with the expression vector to obtain a transgenic insect with a functional human membrane transporter protein.

9. The method as claimed in claim 8, wherein the expression system is GAL4/UAS.

10. The method as claimed in claim 8, wherein the transgenic insect is a Drosophila and the insect comprising the expression system matched with the expression vector is a GAL4 Drosophila line.

11. The method as claimed in claim 14, wherein introducing a selected one of a synthetic compound to be studied and a or natural compound to be studied comprises: introducing a selected one of a synthetic drug or active pharmacological ingredient to be studied and a natural drug or active pharmacological ingredient to be studied.

12. (canceled)

13. The transgenic insect as claimed in claim 1, wherein the human membrane transporter protein is selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 and genetic variants thereof.

14. A method for testing a synthetic or natural compound to be studied with respect to the interaction thereof with a human membrane transporter protein, the method comprising: selecting a human membrane transporter protein from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2, and genetic variants thereof; providing at least one embryo of a transgenic insect with the selected human membrane transporter protein; introducing a tracer substance into the at least one embryo; initially measuring an accumulation of the tracer substance in at least one of 1) cells or tissues of the at least one embryo in which the selected human membrane transporter protein is specifically expressed, and 2) a tissue-specific lumen of the at least one embryo; introducing a selected one of a synthetic compound to be studied and a natural compound to be studied into the at least one embryo by at least one of injection into the at least one embryo and incubation of the embryos in a dissolved form of the selected compound; subsequently measuring, after introducing the selected compound, an accumulation of the tracer substance in at least one of 1) the cells or tissues of the at least one embryo in which the human membrane transporter protein is specifically expressed, and 2) the tissue-specific lumens of the at least one embryo, with the aid of a selected one of a high-spatial-resolution imaging mass spectrometry method, and a fluorescence microscopy; and determining, with the aid of the selected one of a high-spatial-resolution imaging mass spectrometry method and fluorescence microscopy, that the selected compound interacts with an uptake membrane transporter protein when a reduced accumulation of the tracer substance is measured, in the cells or tissues of the at least one embryo in which the human membrane transporter protein is specifically expressed, in the subsequent measurement compared to the initial measurement, the selected compound does not interact with an uptake membrane transporter protein when an unaltered accumulation of the tracer substance is measured, in the cells or tissues of the at least one embryo in which the human membrane transporter protein is specifically expressed, in the subsequent measurement compared to the initial measurement, the selected compound interacts with an efflux membrane transporter protein when a reduced accumulation of the tracer substance is measured, in the tissue-specific lumens of the at least one embryo, in the subsequent measurement compared to the initial measurement, and the selected compound does not interact with an efflux membrane transporter protein when an unaltered accumulation of the tracer substance is measured, in the tissue-specific lumens of the at least one embryo, in the subsequent measurement compared to the initial measurement.

15. The method as claimed in claim 14, wherein the selected compound to be studied is introduced within a period of about 5 minutes to 4 hours after the introduction of the tracer substance.

16. (canceled)

17. The transgenic insect as claimed in claim 1, wherein the human membrane transporter protein is a human membrane transporter protein selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters.

18. The method as claimed in claim 14, wherein the selected compound to be studied is introduced within a period of about 60 min, after the introduction of the tracer substance.

Description

[0075] The figures are described in what follows.

[0076] FIG. 1 shows a schematic representation of an insect embryo with the likewise schematically represented salivary gland as an exemplary specific tissue of an insect embryo (top), and also schematic representations of exemplary embodiments of the use of a transgenic insect according to the present invention (bottom), with an uptake membrane transporter protein (A, B) and with an efflux membrane transporter protein (C, D).

[0077] FIG. 2 shows immunofluorescence images of salivary glands of transgenic Drosophila embryos expressing either the reference sequence of the human SLC22A1/OCT1 protein (SLC22A1 ref) or genetic variants that occur in humans (e.g., SLC22A1 L160F; SLC22A1 G465R).

[0078] FIG. 3A shows the analysis of the uptake of fluorescent ethidium bromide, a transport substrate of OCT1, into the salivary gland of transgenic Drosophila embryos expressing either the reference sequence of the human SLC22A1/OCT1 protein (SLC22A1 ref) or genetic variants that occur in humans (e.g., SLC22A1 L160F; SLC22A1 G465R).

[0079] FIG. 3B shows the ethidium bromide uptake in the epidermis and the salivary gland.

[0080] FIG. 4A shows the uptake of ethidium bromide into the salivary glands of transgenic Drosophila embryos expressing the reference sequence of the human SLC22A1/OCT1 protein.

[0081] FIG. 4B shows the inhibition of the uptake of ethidium bromide with increasing concentration of cimetidine.

[0082] FIG. 5 shows amino acid sequences of exemplary membrane transporter proteins which can be used in the context of the present invention, the following being shown here: OCT1 (SLC22A1) (A) (SEQ ID No. 1), OCT2 (SLC22A2) (B) (SEQ ID No. 2), OATP1B1 (SLCO1B1) (C) (SEQ ID No. 3), OATP1B3 (SLCO1B3) (D) (SEQ ID No. 4), OAT1 (SLC22A6) (E) (SEQ ID No. 5), OAT3 (SLC22A8) (F) (SEQ ID No. 6), MDR1 (ABCB1) (G) (SEQ ID No. 7), BSEP (ABCB11) (H) (SEQ ID No. 8), BCRP (ABCG2) (I) (SEQ ID No. 9), MATE1 (SLC47A1) (J) SEQ ID No. 10), MATE2 (SLC47A2) (K) (SEQ ID No. 11).

EMBODIMENTS OF THE INVENTION

[0083] FIG. 1 depicts, in the top part, an insect embryo 10 (Drosophila), with depiction of the anterior on the left and the posterior on the right. Furthermore, the salivary gland 12 of the insect embryo is drawn in schematically. The salivary gland in the insect embryo consists of a monolayer of epithelial cells that form a lumen. On the basal side thereof, said cells come into contact with blood (hemolymph), and on the apical side, the cells secrete glycoproteins, which are only required during pupation for substrate adhesion by the animal, but not in the embryo or early larval stages.

[0084] The bottom part of FIG. 1 shows enlarged representations of salivary glands as exemplary specific tissue of two different embodiments of transgenic insects: in A and B, a human uptake membrane transporter protein is expressed in the transgenic insect, specifically in a tissue-specific manner, whereas in C and D, an efflux membrane transporter protein is expressed in a tissue-specific manner.

[0085] FIGS. 1 A, B show that the uptake membrane transporter protein is expressed in the basal membrane; the membrane transport protein takes up the tracer, which is supplied by means of injection (A). A simultaneous supply (e.g., injection) of a fluorescent tracer and a substance to be tested, for example a drug, inhibits the accumulation of the fluorescent tracer in the salivary gland (B) when the substance to be studied interacts with the uptake membrane transporter protein.

[0086] FIGS. 1 C, D show that the efflux membrane transporter protein is expressed in the apical membrane; said membrane transport protein transports the tracer, which must be initially taken up into the salivary gland cells, into the lumen of the salivary gland (C). A simultaneous supply (e.g., injection) of a fluorescent tracer and a substance to be tested, for example a drug, inhibits the accumulation of the fluorescent tracer in the lumen of the salivary gland and leads to an increased fluorescence in the salivary gland cells (D) when the substance to be studied interacts with the efflux membrane transporter protein.

[0087] FIG. 2 shows that the membrane transporter proteins OCT1/SLC22A1 reference sequence and its variants L160F and G465R are expressed in the salivary glands of Drosophila melanogaster embryos with the aid of the GAL4/UAS expression system. As is standard, the embryos were fixed for 20 min in 4% formaldehyde, at room temperature and with subsequent repeated washing in phosphate-buffered saline solution. With the aid of an OCT1/SLC22A1-specific antibody and a secondary antibody (Alexa Fluor 568 goat anti-mouse IgG, Invitrogen), the OCT1 proteins in the embryos were detected in standard methods. OCT1/SLC22A1 reference sequence and the variant L160F localize in the basal and lateral cell membrane, whereas the variant G465R can be found in the cytoplasm. What is essentially shown by this experiment is that human membrane transporter proteins are localized in fly embryos in the same way as in human cells.

[0088] FIG. 3 shows that the membrane transporter proteins OCT1/SLC22A1 reference sequence and its variants L160F and G465R are expressed in the salivary glands expressed of Drosophila melanogaster embryos with the aid of the GAL4/UAS expression system. The salivary glands are moreover characterized by the tissue-specific expression of GFP. These living embryos were injected with 0.5 M ethidium bromide. The accumulation of ethidium bromide in the salivary glands was visualized by confocal laser scanning microscopy. The ratio of the signal in the salivary gland cells and in the epidermis cells, which naturally take up ethidium bromide and hence constitute the background, serves for the determination of the efficiency of uptake into the salivary gland cells. Differences in the uptake of ethidium bromide by the variants of, for example, OCT1 reference sequence can thus be established.

[0089] FIG. 4 shows that the membrane transporter protein OCT1/SLC22A1 reference sequence is expressed in the salivary glands of Drosophila melanogaster embryos with the aid of the GAL4/UAS expression system. The salivary glands are moreover characterized by the tissue-specific expression of GFP. Ethidium bromide and cimetidine were simultaneously injected into the ventral side of the embryos. The inhibition of the uptake of ethidium bromide by cimetidine is concentration-dependent. The effect of cimetidine is the same in insects as in human cells.

[0090] As already mentioned above, FIG. 5 shows amino acid sequences of exemplary membrane transport proteins which can be used in the context of the present invention. The amino acid sequences of the following membrane transport proteins are shown here by way of example: OCT1 (SLC22A1) (A), OCT2 (SLC22A2) (B), OATP1B1 (SLCO1B1) (C), OATP1B3 (SLCO1B3) (D), OAT1 (SLC22A6) (E), OAT3 (SLC22A8) (F), MDR1 (ABCB1) (G), BSEP (ABCB11) (H), BCRP (ABCG2) (I), MATE1 (SLC47A1) (J), MATE2 (SLC47A2) (K).