EP3706560A1

EP3706560A1 - Transgenic insect and use of same in methods for testing natural or synthetic substances

Info

Publication number: EP3706560A1
Application number: EP18804246.9A
Authority: EP
Inventors: Elke Schaeffeler; Bernard Moussian; Yiwen Wang; Matthias Schwab; Anne Nies; Bernhard Spengler
Original assignee: Robert Bosch Gesellschaft fuer Medizinische Forschung mbH; Universite de Nice Sophia Antipolis UNSA
Current assignee: Robert Bosch Gesellschaft fuer Medizinische Forschung mbH; Universite de Nice Sophia Antipolis UNSA
Priority date: 2017-11-09
Filing date: 2018-11-06
Publication date: 2020-09-16
Also published as: US20200260700A1; WO2019091966A1; DE102017219981A1

Abstract

The present invention relates to a transgenic insect, the genome of which has at least one first exogenic DNA sequence, which is coded for a human membrane transporter protein, wherein the expression of the first exogenic DNA sequence leads to a functional human membrane transporter protein in the transgenic insect. The invention further relates to uses of the transgenic insect and methods for testing natural or synthetic substances using the transgenic insect.

Description

Transgenic insect and its use in methods of testing natural or synthetic substances Description

The present invention relates to a transgenic insect whose genome has an exogenous DNA sequence. Furthermore, the invention relates to a use of a vector for generating a transgenic insect, as well as methods for testing natural or synthetic substances using the transgenic insect.

State of the art

In drug development, information about clinical factors such as blood and tissue concentrations of drugs and pharmacological agents and their metabolites is of paramount importance. This knowledge essentially helps to better understand the therapeutic efficacy and the occurrence of side effects (Giacomini et al., Nat. Rev. Drug Discov., 2010). In particular, membrane transporter proteins (or membrane-bound transporter proteins) play an important role, since they are able to decisively influence the absorption, distribution and elimination of active substances. These transporter proteins are integral parts of the lipid bilayer of biological membranes. They control or control the transport of numerous substances into the cells (influx) or out of them (efflux). Substance exchange by these membrane transporter proteins can be either passively facilitated or active.

For example, membrane transporters are important determinants of pharmacokinetics, ie the uptake of an active substance (absorption), its distribution in the body (distribution), its biochemical conversion and degradation (metabolism) and its excretion (excretion). For example, it is known that among the top 200 of the

41% of prescription drugs in the US, which are eliminated via the kidney, interact with membrane transporters (Morrissey et al., Annu Rev. Pharmacol Toxicol., 2013). In particular, secretory membrane transporters are important determinants of the disposition of foreign substances (xenobiotics), many of them

prescription drugs. As a result, both the EMA (European Medicines Agency) and the FDA (Food and Drug Administration) are calling for the review of currently eleven relevant ones

Human membrane transporters for possible drug-drug interactions (European Medicines Agency, 2012; FDA draft guidance, drug design studies, data analysis, implications for dosing, and labeling recommendations); 2012). These tests are an essential and compelling part of the preclinical

Drug development process. For example, with in vitro systems, the drug-substrate profile of membrane transporter proteins can be largely predicted. For in vitro models, for example, mammalian cell lines expressing recombinant human membrane transporter proteins are suitable. In culture, they form a single-celled layer, where the permeability and transport of active substances can be investigated. Another established test is the mouse model. However, this is an in vivo test system.

However, the currently used test systems have numerous disadvantages. The systems are time consuming and costly. In particular, the provision of the respective system is very expensive. Furthermore, high operating and maintenance costs are incurred, for example for the large number of mice that have to be previously bred with genetically modified membrane transporters. Not to be neglected are the ethical concerns that must always be considered when carrying out a variety of

Mice experiments is necessary. For this reason, there is an urgent need for fast and cost effective

Test systems to preclinically elucidate the effects of human membrane transporter proteins in the drug development process. The system should be able to overcome the disadvantages prevailing in the prior art. Disclosure of the invention

Against this background, it is an object of the present invention to provide a model or test system with which the aforementioned disadvantages of the prior art can be reduced or avoided altogether. According to the invention, these and other objects are achieved by a transgenic insect whose genome has at least one first exogenous DNA sequence encoding a human membrane transporter protein, expression of the first exogenous DNA sequence resulting in a functional human membrane transporter protein in the transgenic insect.

Further, the object is achieved by the use of a vector for generating transgenic insects, preferably transgenic Drosophila, the vector having a first DNA sequence coding for a human membrane transporter protein, wherein the human membrane transporter protein is in particular a uptake transporter protein or an efflux transporter protein is particularly preferably selected from the group consisting of: OCT1 (gene symbol: SLC22A1), OCT2 (SLC22A2), OATP1B1 (SLC01B1), OATP1B3 (SLC01B3), OAT1 (SLC22A6), OAT3 (SLC22A8), MDR1 (ABCB1 ), BSEP (ABCB1 1), BCRP (ABCG2), MATE1 (SLC47A1), MATE2 (ALC47A2), or a genetic variant of these transporters.

In the present context and in general terms, a "transgenic insect" is an insect whose genetic material has been deliberately altered by genetic methods. In this case, a transgene can be introduced into the insect. According to the invention, a model organism, for example Drosophila, is preferably suitable as the transgenic insect. To obtain such a transgenic insect, vectors containing a specific foreign gene are introduced into a fertilized egg. The resulting offspring are transfected with a certain probability and can then be screened for the foreign gene. The transgenic insect can also be obtained by targeted crossing. Furthermore, the transgenic insect can also be obtained by

Introduction of a transgene into the insect and subsequent cross-breeding with another insect which u.U. genetically modified. In accordance with the present invention, the transgenic insect can be obtained by any of the methods known in the art for altering genes in organisms.

According to the invention, the foreign gene or transgene is a first exogenous DNA sequence which codes for a human membrane transporter protein.

The "human membrane transporter proteins" are those that naturally occur in the human genome. Thus, for example, for the absorption of substances in humans, especially the receiving transporter in the gastrointestinal tract significant. In addition to the nutrients specialized amino acid, peptide, glucose, nucleotide or fatty acid transporters include the organic anion transporter polypeptides (OATP) and the organic cation transporter proteins (OCT). In contrast, efflux transporters in the epithelium of the gastrointestinal tract, such as the ABC proteins (ATP binding cassette proteins), reduce the absorption and thus the oral bioavailability of the active substances binding to and from the cells

be transported out. Membrane transporter proteins such as organic anion transporter polypeptides (OATP), organic cation transporter proteins (OCT), and P-glycoprotein (MDR1 P-gp), bile salt efflux transporter (BSEP), and breast cancer resistance play a key role in liver elimination Protein (BCRP). For elimination by the kidney are crucial above all

Transporter proteins such as organic anion transporters (OAT), organic cation transporter proteins (OCT), and multidrug resistance and toxin extrusion proteins (MATE). Also in other cell membranes, especially biological barriers such as the blood-brain or blood-placental barrier, special transporter proteins are present to regulate mass transfer.

Accordingly, according to one embodiment of the present invention, it is preferred if the human membrane transporter protein is a human uptake transporter protein or a human efflux transporter protein, and in particular a membrane transporter protein selected from the group consisting of: OCT1 (gene symbol: SLC22A1), OCT2 ( SLC22A2), OATP1 B1 (SLC01 B1), OATP1 B3

(SLC01 B3), OAT1 (SLC22A6), OAT3 (SCL22A8), MDR1 (ABCB1), BSEP (ABCB1 1), BCRP (ABCG2), MATE1 (SLC47A1), MATE2 (SLC47A2) or a genetic variant of these transporters. These uptake and efflux transporter proteins, as mentioned above, are being tested for preclinical drug use

Interaction with the respective substances to be examined used. The proteins are organic cation transporter proteins (OCT), organic anion transporter polypeptides (OATP), organic anion transporters (OAT), P-glycoprotein also known as multidrug resistance protein (MDR1),

Bile salt efflux transporter (BSEP), Breast Cancer Resistance Protein (BCRP) as well as Multidrug and Toxin Extrusion Proteins (MATE). The sequences of these and other membrane transporter protein genes are known and, for example, can be viewed at www.genenames.org. For example, according to one embodiment of the present invention, it is preferable to use a membrane transporter protein having an amino acid sequence selected from one of the sequences of Figure 5 or one of them

Sequences encoding DNA sequence. In Figure 5 are exemplary

Amino acid sequences of membrane transporter proteins shown, which may be suitable in the context of the present invention for the described purposes and methods.

In the context of the present invention, "double-stranded DNA sequence coding for a human membrane transport protein" is used herein

Nucleic acid molecule that encodes a human membrane transport protein understood.

A "genetic variant" of a human membrane transporter protein according to the invention and according to the knowledge and experience of a

A specialist in the field of natural protein, which is in his

Amino acid sequence is different from the wild-type or reference sequence and, for example, characterized by the exchange, the deletion or the insertion of one or more amino acids. However, a genetic variant of a human membrane transporter protein preferably still has the same function as the naturally occurring human membrane transporter protein having the wild-type or reference sequence.

The genetic variants can be those variants that occur naturally or are introduced in a targeted manner. The variants may already be on the DNA to be transcribed and thus lead to a variant of the listed proteins.

According to one embodiment, it is preferred if the transgenic insect has at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more exogenous DNA sequences, each coding for a human membrane transporter protein, wherein the expression of the exogenous DNA sequences to functional human membrane transporter proteins in the transgenic insect.

An "exogenous DNA sequence" is understood to mean any DNA sequence which does not naturally occur in the relevant insect, ie without genetic modification of the insect, and which is transferred from another organism, in this case the human, into the genome of the insect and there is stable installed. Correspondingly, "one means exogenous DNA sequence ", that the DNA sequence of another organism (here: of the human) in the insect, ie in the genome of the insect, to the

Expression was installed and there is stable integrated. In a preferred embodiment, the expression of the first exogenous DNA sequence of the human membrane transporter protein in the insect is tissue-specific, in particular salivary gland tissue-specific, intestinal system tissue-specific, tracheid tissue-specific or malpighigeweb-specific. According to the invention, the expression of the first exogenous DNA sequence of the human membrane transporter protein in the transgenic insects is tissue-specific. This allows a more accurate study of human membrane transporters. If the expression occurs only in one tissue, this tissue can be examined separately, so that confounding factors such as overexpression in the entire insect can be avoided.

Furthermore, this embodiment offers the advantage that the expressed human

Membrane transporter protein can be examined tissue-specifically. This allows, for example, the investigation of membrane transporter proteins in the salivary gland, in the intestine, in the trachea or in the Malpighigewebe. Other combinations which are non-tissue specific are also possible.

In particular, salivary gland tissue specificity is advantageous because the target tissue of the salivary gland is not essential at embryonic and early larval stages, so that tissue manipulation, including expression of human proteins and their functional analyzes, may be associated without affecting the fitness and viability of the embryos. In addition, the salivary glandular epithelial cells are polar and therefore resemble human liver or kidney cells expressing mainly clinically relevant human membrane transporters, for example OCT1, OCT2, OATP1B1, OATP1B3 and MDR1.

In a preferred embodiment, the transgenic insect is a fruit fly, in particular one belonging to the genus Drosophila. Drosophila is a genus of the family Drosophila (Drosophilidae). To this genus also belongs the fruit fly Drosophila melanogaster, which is more common Model organism is known in genetics. Especially advantageous for this

Model system is that this type of fly is very easy and cheap to grow. In genetic research, Drosophila is preferably used as a research object because it has a short generation sequence of only about 9 to 14 days, originates from one generation to 400 offspring, each individual has only four pairs of chromosomes and because the species displays many easily recognizable gene mutations. By comparison, model systems based on a mammal are significantly more expensive and expensive.

In a preferred embodiment, the expression of the first exogenous DNA sequence encoding a human membrane transporter protein is under the control of a tissue-specific GAL4 / UAS expression system, in particular under the control of a salivary gland tissue-specific, enteric tissue-specific, tracheid tissue-specific or malignant tissue-specific Furthermore, it is also possible to express the first exogenous DNA sequence using the Q system (Potter et al., Cell 2010).

With the GAL4 / UAS system, a genetic tool is available that allows the expression of foreign genes in specifically selected cells or tissues. Two modules are used for this. First, the baker's yeast GAL4 gene activator (Saccharomyces cerevisiae) is cloned under the control of specific regulatory elements. GAL4 encodes a yeast-specific transcription factor whose expression is under the control of a cell- or tissue-specific promoter from the fly. The other module contains the gene which is responsible for a human

Membrane transporter protein encodes, and is under the control of UAS elements (upstream activating sequences), the target sequences of the GAL4 gene regulator. The specific binding of GAL4 to the so-called UAS ensures the activation of the downstream target gene, which codes for a human membrane transporter protein. By crossing GAL4 lines and those bearing the target gene, such systems can be made.

The GAL4 / UAS system is advantageously applicable to insects, especially Drosophila. According to the invention, GAL4 lines which are tissue-specific

be used to obtain a transgenic insect according to this embodiment. The GAL4 is only expressed in the specific tissues. According to another aspect of the invention, the genome of the transgenic insect comprises at least one second exogenous DNA sequence encoding a fluorescent protein, in particular a fluorescent protein selected from GFP, CFP, YFP, mCherry, dsRed or variants thereof ,

According to this embodiment, it is advantageously possible to directly observe the spatial and temporal distribution in living organisms by the fluorescence of the respective proteins, which can be specifically expressed in the respective tissues. For example, the fluorescent protein can be used as a marker for a protein, for example of the human membrane transporter protein, or to visualize the tissue in which the human membrane transporter protein is expressed. Here, the first exogenous DNA sequence may be linked to the second exogenous DNA sequence. The proteins and thus the biological processes can thus be visualized in vivo, for example by means of fluorescence microscopy or confocal

Laser scanning microscopy.

Suitable fluorescent proteins include the following UV proteins, such as, for example, Sirius, Sandercyanin, shBFP-N158S / L173l; blue proteins such as azurite, EBFP2, mKalamal, mTagBFP2, TagBFP, shBFP; Cyan proteins such as ECFP, Cerulean, m Cerulean3, SCFP3A, CyPet, mTurquoise, mTurquoise2, TagCFP, mTFP1, monomeric Midoriishi cyan, Aquamarine; green proteins such as TurboGFP, TagGFP2, mUKG, Superfolder GFP, Emerald, EGFP, Monomeric Azami Green, mWasabi, Clover, mNeonGreen, NowGFP, mClover3; yellow proteins such as TagYFP, EYFP, Topaz, Venus, SYFP2, Citrine, Ypet, lanRFP-AS83, mPapayal, mCyRFPI; orange proteins such as, for example, Monomeric Kusabira orange, mOrange, mOrange2, ΓΤΙΚΟΚ, mK02; red proteins such as TagRFP, TagRFP-T, mRuby, mRuby2, mRuby3, mTangerine, mApple, mStrawberry, FusionRed, mCherry, mNectarine, mScarlet, mScarlet-1; dark red proteins such as mKate2, HcRed-Tandem, mPlum, mRaspberry, mNeptune, NirFP, TagRFP657, TagRFP675, mCardinal, mStable, mMaroonl, mGarnet2; near IR proteins such as iFP1.4, iRFP713 (iRFP), iRFP670, iRFP682, iRFP702, iRFP720, iFP2.0, mlFP, TDsmURFP, miRFP670; Sapphire-type proteins such as Sapphire, T-Sapphire, mAmetrins; or long Stoke-Shift proteins such as mKeima, mBeRFP, LSS-mKate1, LSS-mKate2, LSSmOrange, CyOFPI, sandercyanine. Variants of the fluorescent protein can be, for example, the redox-sensitive variants of GFP; RoGFP, rxYFP or HyPer could be mentioned here. Further are voltage-dependent GFP variants known as the PROPS or the VSFP. According to the invention, variants of the fluorescent protein are also understood as meaning those proteins which are of natural origin but have been altered on the basis of mutations.

According to a further aspect of the invention, this relates to a method for generating a transgenic insect, the method comprising the following steps:

a) subcloning a first exogenous DNA sequence encoding a human membrane transporter protein into an expression vector to obtain a vector having the first exogenous DNA sequence;

b) introducing the vector obtained in step a) into an insect to obtain a stable strain of a transgenic precursor insect; and c) crossing the transgenic precursor insect with an insect having an expression system adapted to the expression vector to obtain the transgenic insect.

According to this embodiment of the invention, a transgenic insect can be produced quickly and safely. In subcloning, the first exogenous DNA sequence is introduced into another DNA sequence, the expression vector. For this purpose, the exogenous DNA sequence and the expression vector can be cut specifically with the aid of restriction enzymes in order to obtain so-called complementary "sticky-ends" or "blunt" blunt-ends, which can then be ligated together, so that the desired expression vector The ligation can be carried out with the aid of DNA ligases The introduction of the first exogenous DNA sequence into the expression vector can also be carried out by other methods, for example the In-Fusion Cloning System (Takara Bio USA Inc.) ,

According to the invention, plasmids, cosmids or YACs and modified viruses can be used as expression vectors; preferably a plasmid vector is used.

For example, a UAS-appropriate vector can be used. This allows the application of the GAL4 / UAS system in the generation of a transgenic insect. Furthermore, the vector may have a gene sequence specific to a particular gene

Phenotypes coded. As a result, it is advantageously possible to carry out a subsequent selection of transgenic precursor insects on the basis of the phenotype. The pronounced phenotype serves as a selection marker. Mutations leading to one For example, eye color, body color, morphology of the eye, limb morphology, including the wings, body bristles, body colors, etc., may be included. In a preferred embodiment, the human membrane transporter gene may be incorporated into the attP Attachement site in the Expression vector can be introduced so that, for example, a pUASTattB expression vector is obtained.

In the next step, step b), the introduced vector is introduced into an insect. The vector can be microinjected into an insect embryo. The stable strain of a transgenic precursor can then be associated with some

Probability to be obtained. If the expression vector has a

Having selection marker, the transgenic precursor insects can be identified by this and sorted out accordingly. For example, the eye color or the body color can be used as a selection marker.

In the final step of the method of generating a transgenic insect, the precursor insect is crossed with another insect. The two insects differ in their genome. While the precursor insect already contains the gene of the human membrane transporter protein, the other insect does not have this gene.

Preferably, the latter insect is a tissue specific line whose expression system is matched to that of the precursor insect.

The correctly expired cross can be confirmed by expression in the offspring by reverse transcriptase PCR. Membrane localization of human transporter proteins in the specific tissues can be accomplished by

Standard immunostaining techniques are verified with validated primary antibodies. For example, immunofluorescence images may be confocal

Laserscanningmikroskopie be obtained.

According to a further embodiment of the method, the expression system

GAL4 / UAS.

Advantageously, the expression vector in which the first exogenous DNA sequence encoding a human membrane transporter protein is subcloned is suitable for use in a GAL4 / UAS expression system. Preferably, the Expression vector on a UAS. By crossing the precursor, which has the expression vector with the UAS, with a GAL4 line, which also

tissue specific, the expression of the human membrane transporter proteins can be controlled easily and safely.

According to a further embodiment of the method, the transgenic insect is a Drosophila and the insect used in step c) which has an expression system adapted to the expression vector is a GAL4 Drosophila monkey. This embodiment is preferred because many GAL4 Drosophila Umen are already known and are suitable for crossing within a UAS system. As GAL4 lines, for example, P (fkh-Gal4), P (GawB) 34B or P (GawB) C-765 can be used. According to a further aspect of the invention, this relates to a use of a transgenic insect for testing synthetic or natural compounds, in particular medicaments and pharmacologically active substances.

With this embodiment, by the transgenic insect a novel and

cost-effective in-situ assay system, which allows

Interaction between human membrane transporter proteins and synthetic or natural compounds, in particular drugs and pharmacological agents to investigate. Thus, the transgenic insects can be used for pharmacological screening. The interaction can be investigated by various imaging techniques, for example by means of fluorescence spectroscopy.

According to a further embodiment, the interaction by means of

transporter-specific fluorescent tracer substrates are examined. These can be injected into embryos of the transgenic insects, which are transparent and express the human membrane transporter protein. Subsequently, the

Incorporation of the fluorescent tracer substrate in the cells or tissues in which the human membrane transporter protein is specifically expressed, for example, be observed by fluorescence microscopy. It can be compared how the respective fluorescent tracer substrates behave in the absence or presence of the respective test substances. In this context, substances that interact with a receiving transporter can lead to reduced accumulation of the fluorescent tracer and thus to reduced fluorescence within the cell. On the other hand, substances that interact with an efflux transporter may increase

Fluorescence in the respective tissue cells and a reduced fluorescence signal in the tissue lumen lead.

It is particularly advantageous in this system that within 30 minutes up to 100 fly embryos can be injected and screened. Furthermore, it is particularly advantageous that the model system is a very small insect embryo (0.5 mm), so that this system is used for automated analysis of the

Drug interaction with the membrane transporter proteins is suitable.

The new assay system based on the transgenic insects may be able to replace existing mouse models, creating a model that is ethically safe.

According to a further embodiment of the invention, the compound is tested for its interaction with human membrane transporter proteins, wherein the human membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP , MATE1, MATE2 or a genetic variant transporter.

According to a further aspect of the invention, this relates to a use of a transgenic insect for testing the function of human

Membrane transporter proteins, wherein the human membrane transporter protein

is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters.

It is understood that the above statements made with regard to

Membrane transporter proteins and the transgenic insect are also applicable to the described use here described as well as for methods that are carried out according to the invention and as described below. In addition to the interaction with human membrane transporter proteins, the function of human membrane transporter proteins can also be investigated in the same model system. The function of human membrane transporter proteins is preferably investigated in situ. In this case, the respective substances to be investigated are preferably injected into the insect embryos, these being incorporated, for example, in gelatine. Subsequently, thin sections (typically 20 μm thick) of the respective insect are optionally produced in a cryostat in a cryostat. Subsequently, a matrix substance is applied to the embryos or their sections by pneumatic spraying. This matrix consists of small organic molecules that absorb the introduced laser energy and provide for the desorption and ionization of the analytes. The respective substances to be examined can be resolved spatially in the tissue of the embryos directly by the use of a

Atmospheric pressure scanning Microprobe matrix-assisted laser desorption ionization (SMALDI) ion source coupled with, for example, an orbital trap mass spectrometer can be measured. The obtained images and data can be used to determine the spatial distribution and quantity of the examined

Determine substances in the respective tissues.

According to another aspect of the invention, it relates to a method of assaying a synthetic or natural compound to be tested for its interaction with human membrane transporter proteins, wherein the human

Membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters, the method comprising the following sequential steps:

a) providing embryos of a transgenic insect;

b) introducing a first tracer substance into the embryos and measuring the accumulation of the tracer substance in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed; and / or measuring the accumulation of the tracer substance in the tissue-specific lumen of the embryos;

c) introduction of a synthetic or natural to be examined

Compound in the embryos by injection into the embryos or incubation of the embryos in the dissolved compound;

d) measuring the accumulation of the tracer substance in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed; and / or measuring the accumulation of

Tracer substance in the tissue-specific lumen of the embryos; and e) determination by means of a reduced accumulation of the tracer substance or an unchanged accumulation of the tracer substance in the cells or tissues of the embryos in which the human

Membrane transporter protein is specifically expressed whether the compound interacts with a host membrane transporter protein or not; and / or determination, based on a decreased accumulation of

Tracer substance or an unchanged accumulation of

Tracer substance in the tissue-specific lumen of the embryo, whether the compound interacts with an efflux membrane transporter protein or not.

According to a preferred embodiment of this method, the tissue-specific lumen of the embryos is selected from the salivary gland lumen, intestinal lumen, the lumen of the Malpighi vessels, and the tracheal lumen of the embryos.

According to a further preferred embodiment, the measuring takes place

Accumulation of the tracer substance in the tissue-specific lumen of the embryos and / or the determination, by means of a reduced accumulation of the tracer substance or an unchanged accumulation of the tracer substance in the tissue-specific lumen of the embryos, whether the compound interacts with an efflux membrane transporter protein or not, using the high-spatial resolution -bildgebenden

Mass spectrometry or by fluorescence microscopy.

According to a preferred embodiment, the introduction of the compound to be examined in step b) takes place in a period of about 5 minutes to 4 hours, preferably about 60 minutes after the simultaneous introduction of the tracer substance and a compound to be examined.

According to a further aspect of the invention, this relates to a method for directly testing the function of human membrane transporter proteins, wherein the human membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP , BCRP, MATE1, MATE2 or a genetic variant of these transporters, the method comprising the following sequential steps: a) providing embryos of a transgenic insect;

b) introducing a synthetic or natural compound into the

Embryos by injection into the embryos or incubation of the embryos

Embryos in the dissolved compound; and

c) measuring the presence and / or amount of the introduced in step b)

Compound in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed.

In this case, according to a preferred embodiment of this method, the accumulation of a synthetic or natural compound in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed, in particular in the cells of the salivary glands or other cell types of the embryos; and / or measuring the accumulation of the synthetic or natural compound in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed, in particular the

Salivary gland lumen or in the lumen of intestine, Malpighi vessels, embryonic trachea, using high-resolution and / or high-resolution imaging mass spectrometry or by fluorescence microscopy. Advantages of the invention

The present invention is not in its application to the details of the preparation and arrangement of the components shown in the description or in the

Drawings are limited. The invention allows others

Forms of training and can be carried out in various ways. Also, the phraseology and terminology used herein are for descriptive purposes and are not to be considered as limiting. The use of "including," "comprising," or "having," "including," "including," and variations thereof is intended to encompass herein after, as well as equivalents thereof and additional thereto.

The present invention will be further illustrated by the following examples, which in no way are to be considered as further limiting. The entire contents of all references cited in the present application (including references, issued patents, published patent applications and co-pending patent applications) are hereby expressly incorporated herein by reference. in the Trap of a conflict is the present description, including any

Definitions herein, priority.

The following is the description of the figures.

FIG. 1 shows a schematic representation of an insect embryo with the salivary gland likewise schematically represented as an exemplary specific tissue of an insect embryo (top), as well as schematic representations of an example

Embodiments of the use of a transgenic insect according to the present invention (below), once with a uptake membrane transporter protein (A, B), and once with an efflux membrane transporter protein (C, D).

Figure 2 shows immunofluorescence images of salivary glands of transgenic Drosophila embryos expressing either the human SLC22A1 / OCT1 human reference sequence (SLC22A1 ref) or various human genetic variants (e.g., SLC22A1 L160F; SLC22A1 G465R).

Figure 3A shows the analysis of the uptake of fluorescent ethidium bromide, a transport substrate of OCT1, into the salivary gland of transgenic Drosophila embryos containing either the human SLC22A1 / OCT1 human reference sequence (SLC22A1 ref) or various human variant genetic variants (SLC22A1 ref). eg SLC22A1 L160F; SLC22A1 G465R).

Fig. 3B shows the ethidium bromide uptake of the epidermis and salivary gland.

Figure 4A shows the ethidium bromide uptake in the salivary glands of transgenic Drosop / / / 7a embryos expressing the reference sequence of the human SLC22A1 / OCT1 protein. Fig. 4B shows the inhibition of Ethidiumbromidaufnahme with increasing

Concentration of cimetidine.

Figure 5 shows amino acid sequences of exemplary membrane transporter proteins which may be used in the present invention, the following are shown: OCT1 (SLC22A1) (A) (SEQ ID NO: 1), OCT2 (SLC22A2) (B) (SEQ ID No. 2), OATP1B1 (SLC01B1) (C) (SEQ ID NO: 3), OATP1B3 (SLC01B3) (D) (SEQ ID No. 4), OAT1 (SLC22A6) (E) (SEQ ID No. 5), 0AT3 (SLC22A8) (F) (SEQ ID No. 6), MDR1 (ABCB1) (G) (SEQ ID No. 7), BSEP (ABCB1 1) (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

In Fig. 1, an insect embryo 10 (Drosophila) is shown in the upper portion, with anterior left and posterior right. Furthermore, the salivary gland 12 of the insect embryo is schematically drawn. The salivary gland in the

Insect embryo consists of an epithelial cell monolayer, which form a lumen. At the basal side, these cells come into contact with blood (hemolymph), and on the apical side the cells secrete glycoproteins, which are required for the substrate adhesion of the animal only during pupation, but not in the embryo or early larval stages.

In the lower part of FIG. 1, enlarged representations of salivary glands are shown as exemplary specific tissues of two different embodiments of transgenic insects: In A and B, a human uptake membrane transporter protein is expressed in the trans to insect, specifically tissue-specific, whereas in C and D, a human uptake membrane transporter protein is expressed Efflux membrane transporter protein is expressed tissue-specifically.

In Figures 1 A, B it is shown that the uptake membrane transporter protein is expressed in the basement membrane; the membrane transport protein picks up the tracer, which is injected (A). Simultaneous delivery (eg, injection) of a fluorescent tracer and a substance to be tested, for example, a drug, inhibits accumulation of the fluorescent tracer in the salivary gland (B) when the substance to be assayed interacts with the uptake membrane transporter protein.

In FIGS. 1 C, D it is shown that the efflux membrane transporter protein in the

Apical membrane is expressed; This membrane transport protein transports the tracer, which must first be taken up into the salivary gland cells, into the lumen of the salivary gland (C). Simultaneous delivery (eg 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 leads to increased fluorescence of the salivary gland cells (D) when the substance to be studied interacts with the efflux membrane transporter protein. In Fig. 2 it is shown that by means of the GAL4 / UAS Expressionssytems the

Membrane transporter proteins OCT1 / SLC22A1 reference sequence and its variants L160F and G465R are expressed in the salivary glands of Drosophila me / anogasfer embryos. The embryos were fixed by default in 4% formaldehyde for 20 min at room temperature followed by multiple washes in phosphate buffered saline. With the help of an OCT1 / SLC22A1-specific antibody and a secondary antibody (Alexa-Fluor 568 goat anti-mouse IgG, Invitrogen), the OCT1 proteins were detected in the embryos in standard procedures. OCT1 / SLC22A1 reference sequence and variant L160F localize in the basal and lateral cell membrane, whereas variant G465R is found in the cytoplasm. in the

Essentially, this experiment shows that human membrane transporter proteins in

Flying embryos are localized as in human cells.

In Fig. 3 it is shown that by means of the GAL4 / UAS Expressionssytems the

Membrane transporter proteins OCT1 / SLC22A1 Reference sequence and its variants L160F and G465R expressed in the salivary glands are expressed by Drosophila melanogaster embryos. The salivary glands are also due to the

tissue-specific expression of GFP. These live embryos were injected with 0.5 μΜ 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, the

Absorbing ethidium bromide naturally and thus providing the background serves to determine the efficiency of uptake into the salivary gland cells.

Differences in the uptake of ethidium bromide by the variants eg of OCT1 reference sequence can thus be determined.

In Fig. 4 it is shown that by means of the GAL4 / UAS Expressionssytems the

Membrane transporter protein OCT1 / SLC22A1 reference sequence is expressed in the salivary glands of Drosophila me / anogasier embryos. The salivary glands are also characterized by the tissue-specific expression of GFP. Ethidium bromide and cimetidine were injected simultaneously into the ventral side of the embryos. The inhibition Ethidium bromide uptake by cimetidine is concentration dependent. The effect of cimetidine is the same in insects as in human cells.

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 (SLC01B1) (C), OATP1B3 (SLC01B3) (D), OAT1 (SLC22A6) (E), OAT3 (SLC22A8) (F), MDR1 (ABCB1) (G), BSEP (ABCB1 1) (H), BCRP (ABCG2) (I), MATE1 (SLC47A1) (J), MATE2 (SLC47A2) ( K).

Claims

claims

1 . A transgenic insect whose genome has at least one first exogenous DNA sequence encoding a human membrane transporter protein, wherein expression of the first exogenous DNA sequence results in a functional human

Membrane transporter protein in the transgenic insect leads.

2. Transgenic insect according to claim 1, characterized in that the expression of the first exogenous DNA sequence of the human membrane transporter protein in the insect is tissue-specific, in particular salivary gland tissue-specific,

Intestinal system tissue-specific, tracheo-tissue-specific or malpigious-tissue-specific.

3. Transgenic insect according to claim 1 or 2, characterized in that the transgenic insect is a fruit fly, in particular one belonging to the genus Drosophila.

4. Transgenic insect according to one of claims 1 to 3, characterized in that the human membrane transporter protein is a human uptake transporter protein or a human efflux transporter protein, in particular 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.

5. Transgenic insect according to one of claims 1 to 4, characterized in that the expression of the first exogenous DNA sequence responsible for a human

Membrane transporter protein, is under the control of a tissue-specific GAL4 / UAS expression system, in particular under the control of a salivary gland tissue-specific, enteric tissue-specific, tracheid tissue-specific or malignant tissue-specific GAL4 / UAS expression system.

6. Transgenic insect according to one of claims 1 to 5, characterized in that its genome has at least one second exogenous DNA sequence which codes for a fluorescent protein, in particular for a fluorescent protein which is selected from GFP, CFP, YFP, mCherry, dsRed or variants of these.

7. Use of a vector for generating transgenic insects, preferably transgenic Drosophila, wherein the vector has a first DNA sequence which codes for a human membrane transporter protein, wherein the human

Membrane transporter protein, in particular a uptake transporter protein or an efflux transporter protein, is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these Transportation.

8. A method of generating a transgenic insect according to any one of claims 1 to 6, said method comprising the steps of:

a) Subcloning of a first exogenous DNA sequence encoding a human

Membrane transporter protein encoded in an expression vector to obtain a vector having the first exogenous DNA sequence;

b) introducing the vector obtained in step a) into an insect to obtain a stable strain of a transgenic precursor insect; and

c) Crossing of the transgenic precursor insect with an insect that binds to the

Expression vector adapted expression system, to obtain the transgenic insect.

9. The method according to claim 8, characterized in that the expression system is GAL4 / UAS.

10. The method according to claim 8 or 9, characterized in that the transgenic insect is a Drosophila, and the insect used in step c), the one to the

Expression vector adapted expression system is a GAL 4-Drosophila line.

1 1. Use of a transgenic insect according to any one of claims 1 to 6 for the testing of synthetic or natural compounds, in particular of drugs and pharmacological agents.

12. Use according to claim 1 1, characterized in that the compound is tested for their interaction with human membrane transporter proteins, wherein the human membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1 B1, OATP1 B3, OAT1, OAT3 , MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters.

Use of a transgenic insect according to any one of claims 1 to 6 for assaying the function of human membrane transporter proteins, wherein the human

Membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters.

14. A method for testing a synthetic or natural compound to be tested for its interaction with human

Membrane transporter proteins, wherein the human membrane transporter protein

is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1B1, OATP1B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters, the method comprising the following

having successive steps:

a) providing embryos of a transgenic insect;

c) introduction of a synthetic or natural to be examined

Tracer substance in the tissue-specific lumen of the embryo, using high-resolution, imaging mass spectrometry or fluorescence microscopy; and

e) Determination by means of a reduced accumulation of the tracer substance or an unchanged accumulation of the tracer substance in the cells or tissues of the embryos in which the human

Membrane transporter protein is specifically expressed whether the compound interacts with a host membrane transporter protein or not; and / or determination, based on a reduced accumulation of the tracer substance or an unchanged accumulation of the Tracer substance in the tissue-specific lumen of the embryo, whether the compound interacts with an efflux membrane transporter protein or not, using a high-spatial resolution, imaging

Mass spectrometry or by fluorescence microscopy.

15. The method according to claim 14, characterized in that the introduction of the compound to be examined in step b) in a period of about 5 minutes to 4 hours, preferably about 60 minutes, after the introduction of the first tracer substance and a to examining compound takes place.

16. A method for testing the function of human membrane transporter proteins, wherein the human membrane transporter protein is particularly preferably selected from the group consisting of: OCT1, OCT2, OATP1 B1, OATP1 B3, OAT1, OAT3, MDR1, BSEP, BCRP, MATE1, MATE2 or a genetic variant of these transporters, the method comprising the following sequential steps:

a) providing embryos of a transgenic insect according to any one of claims 1 to 6;

b) introduction of a synthetic or natural compound into the embryos by injection into the embryos or incubation of the embryos in the dissolved compound; and

c) measuring the presence and / or amount of the introduced in step b)

A compound in the cells or tissues of the embryos in which the human membrane transporter protein is specifically expressed using high resolution, imaging mass spectrometry or by means of

Fluorescence microscopy ..