Gene library and a method for producing the same

Abstract
The invention relates to a method for producing a gene library, preferably a cDNA gene library and to a gene library that can be obtained by said method.
Description


[0001] The present invention relates to a method for producing a gene library, preferably a cDNA gene library, and to a gene library that can be obtained by said method.


[0002] A gene library constitutes a collection of recombinant DNA molecules in the form of e.g. bacteria/plasmid clones, phage lysates, etc. In the ideal case, the respective vector inserts represent the complete genetic information for example of a special organism or tissue, whereat the probability to find a specific gene within such a collection is dependent on the size of the insert, the total genome size and the copy number of the respective sequence element, e.g. the gene, in the genome. cDNA libraries offer the advantage, that they only comprise genes, which are actually expressed and which are free of the intron parts. Moreover, cDNA libraries can be relied upon when investigating the gene expression in specific cells and/or tissues in response to specific factors, e.g. the status of differentiation. However, the problem, that the respective whole gene or the desired parts of the gene are not present within the gene library is frequent to arise in consequence of the primers used for cDNA synthesis from the mRNA template.


[0003] In any case, the currently used methods to produce cDNA libraries are laborious, hardly efficient and impaired by a series of further disadvantages, which are discussed in the following. It should be mentioned, that the major difficulty in using the ordinarily produced cDNA libraries for investigating gene expression by means of microarrays is to be found both in un-specific cross-reactions of the spotted cDNA clones—irrespective of being obtained as plasmids or PCR amplification products—when hybridizing with the RNA-samples, and in a poor signal to noise ratio. Both factors may lead to a complete misinterpretation of hybridization results and thus of the whole experiment. Specifically, these problems may be due to various reasons:


[0004] a) Conserved domains in the coding sequences of particular members of a gene family lead to a non-specific cross-reaction.


[0005] b) Since in general one has to use amplification primers, which correspond to the employed vector in order to produce the PCR-amplified products, normally vector parts are co-amplified. This can lead both to an unspecific reaction and to a significant deterioration of the signal to noise ratio.


[0006] c) The commonly employed amplification primers display a relatively low melting temperature (Tm). Accordingly, the amplification reaction has to be accomplished at a rather moderate annealing temperature. The risk of an non-specific binding of the amplification primers and in consequence the risk of a non-specific amplification is thus quite high.


[0007] d) The different cDNA-molecules display poly(A)-tails of variable length, which may lead both to an increased background and to a non-specific hybridization reaction.


[0008] e) When using the ordinarily produced cDNA libraries for the production of microarrays, the cDNA-inserts are in general present in a non-orientated fashion within the polylinker of a vector system. It is thus impossible to spot only the “antisense”-strand by using just one modified PCR-Primer. This also adds to the significant deterioration in the signal to noise ratio.


[0009] f) The extremely variable sizes of the different cDNAs, which are ranging between 500 bp and >5 kb, cause very inhomogeneous yields in the PCR. To allow a representative hybridization experiment however, the cDNA-microarray must be spotted with homogeneous, equimolar amounts of the distinct clones.


[0010] Thus, the present invention is primarily based on the technical objective to provide a method to produce a gene library, which avoids the above-mentioned problems.


[0011] This technical objective was achieved by providing the embodiments, which are characterized in the claims.


[0012] It has surprisingly been found, that a gene library can be produced by means of the method described in the following example, whereat this gene library offers a series of major advantages in comparison to the common methods, in particular in respect to the generation of cDNA libraries, which are optimized for the investigation of gene expression by means of microarrays. The characteristics of a collection of clones produced by the method according to the invention thus prove to be advantageous both in the production of the cDNA-microarrays itself and in the actual employment of the microarrays in hybridization experiments.


[0013] Since the cDNA-inserts of the recombinant bacterial clones normally have to be amplified by the polymerase chain reaction (PCR) for producing the cDNA-microarrays, high and homogeneous yields in the PCR are favorable. The amplified products should furthermore be utmostly free of contaminations by other amplified elements, which are due to a non-specific annealing of the primer during PCR. For this reason, the method according to the invention employs specific primer binding sites being ligated at flanking positions of the cDNA-fragments, whereat the sequences of these primer binding sites show an increased GC-contents, which allows to increase the primer binding temperature (annealing temperature) in the PCR. The rise of non-specific background products is significantly less probable in case of using increased annealing temperatures.


[0014] In comparison to other cDNA libraries used for microarray applications, the cDNA libraries obtainable by the method according to invention provide the additional advantage, that a very homogeneous size distribution of about 200-600 bp can be achieved, which is not at least also responsible for the homogeneous yields in PCR amplification. Moreover, the homogeneous size distribution of the cDNAs—when producing microarrays at a given concentration of the initial solution—allows to apply nearly equimolar amounts e.g. on a glass carrier. A laborious individual adjustment of the molar concentration of the spotting solution is thus avoided.


[0015] A further advantage of the cDNA-fragments obtainable by the method according to the invention, which both affects the production of microarrays and the hybridization results, is the directed/orientated cloning of the cDNAs. In the vector, these cDNAs are present in a uniformly defined orientation, such that—by means of specific PCR-primer modification and suitable coupling chemistry—the “sense strand” alone can be selectively coupled to the employed glass carrier. In standard hybridization with “antisense”-labeled cDNA, such “single strand”-microarrays provide a significantly improved sensitivity in respect to their binding behavior.


[0016] A whole series of further advantages directly refers to the behavior of the cDNA-fragments contained within the gene libraries according to the invention when used in complex hybridization for elucidating the level of gene expression. Background hybridizations, which reduce the sensitivity of the system, or non-specific hybridizations, which feign false positive results, are not desired and may negatively influence the test result in case of using inappropriate cDNA-fragments. For this reason, the primer binding sites for PCR amplification of the cDNAs directly flank the cDNA-insert in the method according to the invention. Thus, no non-specific vector parts, which could give reason for an undesirable background hybridization, are amplified; The previous collections of cDNA-clones frequently lead to the generation of PCR products containing larger portions (>100 bp) of vector sequences and thus reduce the specificity of the cDNA amplification product.


[0017] A major advantage of the method according to the invention moreover is the generation of cDNA-fragments, which are originally derived from the 3′-untranslated region of the mRNA. These sequence sections mostly develop in a very divergent manner due to the missing selective pressure in phylogenesis, such that even closely related members of a gene family can be clearly distinguished in a hybridization analysis for gene expression. Cross-hybridizations between evolutionary strongly conserved sequence sections preferably occur in the coding sequence portions in conventional cDNA-libraries. Due to the 3′-selection of cDNAs in combination with the stringent size selection of 200-600 bp, primarily the 3′-untranslated regions of transcripts are represented in the cDNA libraries obtainable by the method according to the invention, which permit an increased specificity of hybridization in microarray applications. The homopolymeric poly(A)-tails marking the 3′-end of a transcript, which are frequently present in conventional cDNA libraries, are removed in the course of the method according to the invention. Poly(A)-tails of larger sizes on a microarray reduce the specificity of hybridization and thus cause a deterioration of the signal to noise ratio.


[0018] The method according to the invention is mainly based on the following steps:


[0019] (a) Starting from mRNA, synthesizing the first cDNA-strand by using a primer, which provides at its 5′-end a first partner of a binding pair, preferably bound in a covalent form, whereat this first binding partner has an affinity to a second binding partner of the binding pair. Suitable binding pairs are familiar to the expert and comprise e.g. biotin/streptavidin, biotin/avidin, antigen/antibody, etc. The primer moreover contains at its 3′-end a poly(dT)-sequence of sufficient length, which is complementary the poly(A)-tail of the mRNA, and—between the 5′-end being coupled to the first binding partner and the poly(dT)-sequence—a sequence, which as a double strand provides a recognition site for a type II restriction enzyme (e.g. BpmI), thereby allowing to later cut off the poly(dT)-sequence or a part thereof from the cDNA. Preferably, the primer is a mixture of primers, which provide the sequence element 5′-VN-3′ at the 3′-side of the poly(dT)-sequence, whereat V is A, C or G and N is A, C, G or T;


[0020] (b) second strand synthesis under common conditions, e.g. by (1) degrading the RNA of the DNA/RNA-hybrid with RNAse H, (2) generating the complementary strand with DNA-Polymerase I and (3) linking the open Okazaki-fragments by means of ligase;


[0021] (c) fragmentation of the DNAs of step (b) by means of common methods, preferably by using random fragmentation, whereat fragmentation by means of sonication is preferred;


[0022] (d) isolation of fragments with the desired range of length by means of common methods if favorable, e.g. by using agarose gel electrophoresis and isolating the desired fragments from the gel. Preferably isolated are fragments in the range of 100 to 1000 bp, more preferred in the range of 200 to 600 bp. In the following, the ends of the DNA-fragments are preferably enzymatically transformed into blunt ends, i.e. the overhangs of the 3′-end or of the 5′-end are removed, for example by using Pwo-DNA-Polymerase or Pfu-DNA-Polymerase;


[0023] (e) binding of the fragments, which correspond to the 3′-region of the mRNA to the second binding partner via the first binding partner, whereat the second binding partner is coupled to a solid carrier. Suitable solid carriers are familiar to the expert; they comprise e.g. paramagnetic pearls. The fragments, which remain unattached (fragments without the first binding partner) are then removed by washing.


[0024] (f) an adaptor molecule is ligated to the DNA-fragment end opposing the biotinylated end of the DNA-fragment by means of common methods. This adaptor in its 5′-overhanging sequence contains a first restriction site for a restriction enzyme, which preferably cleaves in such a way, that DNA-ends with sequence overhangs are generated (to this: also see the exemplified adaptor in the following example);


[0025] (g) a PCR is performed with the fragments still being coupled to the solid carrier, whereat the following primers are used: The 5′-primer is a primer, which mainly corresponds to the sequence of the overhanging adaptor end (or a part thereof). The 3′-primer at its 5′-end is connected to a first binding partner of a binding pair, which can be identical with or different to the binding partner of step (a), and has a sequence mainly corresponding to the sequence of the primer used for first strand synthesis including the restriction site for a type II restriction enzyme. Thus, the 3′-primer in the PCR causes the synthesis of a strand, the sequence of which largely corresponds to that of the first cDNA-strand. The 5′-primer anneals to this strand, thereby leading to the synthesis of the complementary strand under introduction of the restriction site of the adaptor;


[0026] (h) after termination of the PCR and preferable purification of the amplified products, a digest is performed with the suitable type II restriction enzyme, whereby the poly(dT)-tails are cut off. Preferably, two to four dT-residues are retained at the fragments. The cleaved poly(dT)-tails are then removed from the fragments now being without a binding partner by binding via their first binding partner to the second binding partner, which is coupled to a solid carrier;


[0027] (i) the PCR-fragments disconnected from the poly(dT)-tails (PCR-fragments without binding partner) are preferably again tested in respect to their length and fragments within the desired range of length (see step d) are isolated. Thereafter, an adaptor is ligated to that fragment end, the poly(dT)-tail of which has been removed. This adaptor preferably provides a free d(TT) at one end and is thus able to hybridize to the d(AA)-overhang of the preferred PCR-fragments of step (h). At its other overhanging end, the adaptor provides a recognition site for a second restriction enzyme, which preferably produces ends with sequence overhangs, and whereat the recognition site preferably is different from the first recognition site of the first adaptor molecule in step (f); for a possible design of this adapter we refer to the following example; and in the last step the fragments of step (i) are cut with the restriction enzyme for the first recognition site (the restriction cleavage site for the second restriction enzyme is already “open”) and the fragments are ligated into a vector, which has been cut in a parallel manner. Preferably, the fragments are inserted by ligation in a defined orientation. It is not obligate, that this vector is already the vector used for the actual gene library, but it may also be an “intermediate” vector. By means of this vector, the ligation mixture is amplified in an appropriate host, e.g. E. coli by means of standard protocols (see e.g. Maniatis et al., 1989, “Molecular Cloning: A Laboratory Manual”, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.). After the in vivo-amplification, the vector-DNA is isolated from the transformants and introduced into the final host in order to establish the final gene library (see e.g. the review article in “Current Protocols in Molecular Biology”, second ed., 1988, eds.: Ausubel et al., Greene Publish Assoc. & Wiley Interscience, chapter 13).


[0028] Thus, the present invention refers to a method for producing a gene library, whereat said method comprises the following steps:


[0029] a) Production of a double-stranded cDNA from a mRNA-population, wherein a specific primer is used for the synthesis of the first cDNA-strand, this primer providing at its 5′-end a first binding partner of a binding pair with an affinity to a second binding partner of the binding pair, this primer further containing at its 3′-end a poly(dT)-sequence, which is complementary the poly(A)-tail of the mRNA, and furthermore containing a sequence, which as a double strand comprises a recognition site for a type II restriction enzyme, whereat said sequence is located between the 5′-end coupled to the first binding partner and the poly(dT)-sequence;


[0030] b) fragmentation of the cDNA obtained in step (a);


[0031] c) if desired, isolation of fragments of step (b) having the desired length;


[0032] d) if desired, providing the (isolated) fragments with blunt ends;


[0033] e) binding the fragments of the respective previous step to a second binding partner, which is coupled to a solid carrier;


[0034] f) ligating a double-stranded adaptor molecule to the fragment end opposing the end connected to the first binding partner, whereat the adaptor molecule contains a restriction site for a second restriction enzyme;


[0035] g) Performance of an in vitro-amplification of the fragments being bound to the carrier by means of a pair of primers, whereat the first primer of the primer pair is mainly complementary to a strand of the adaptor molecule, and whereat the second primer of the primer pair at its 5′-end provides a first binding partner with an affinity to a second partner of a binding pair, and the sequence of which mainly corresponds to the sequence of the primer used in step (a) or a sequence complementary thereto;


[0036] h) if desired, cleaving the amplification products of step (e) with a type II restriction enzyme, which recognizes the restriction site introduced in step (a);


[0037] i) if desired, ligating an adaptor molecule to the 5′-end of the PCR-products obtained after cleaving with the type II restriction enzyme according to the previous step;


[0038] j) if desired, cleaving the products of step (i) by means of a second restriction enzyme, which recognizes the restriction site introduced in step (f), whereat the steps (i) and (j) may also be performed in a reverse order; and


[0039] k) ligating the products of the respective previous step into a vector.


[0040] The chronological order of single or several steps of the method according to the invention does not have to be performed categorically in the presented order, but may potentially also be varied. Moreover, the mentioned steps c), d) and h)-j) are to be understood as optional. The production of the first and second cDNA-strand is accomplished by means of standard methods under employment of common enzymes and buffers. The expert also is familiar with suitable methods for the isolation of RNA from a sample, wherein the mRNA preferably is enriched e.g. by means of an oligo(dT)-column. The length of the poly(dT)-sequence of the primers used for the first strand synthesis is not critical; it usually ranges between 15 to 30 residues, whereat 18-mers to 21-mers are preferred. Preferably used for the first strand synthesis is a mixture of primers, which provide the sequence 5′-VN-3′ at the 3′-side of the poly(dT)-sequence, whereat V can be A, C or G and N can be A, C, G or T.


[0041] The expert also knows about suitable binding partners of the binding pair, e.g. antigen/antibody, antibody/digoxigenin, biotin/avidin and biotin/streptavidin, whereat biotin/avidin and biotin/streptavidin are preferred.


[0042] The recognition site of the type II restriction enzyme may be any recognition site of a type II restriction enzyme, e.g. BpmI, AlwI, BpsI, BpvI, Bci VI, BsaI, Bse RI, BsgI, Bsm A1, Earl, EciI, FokI, HgaI, HphI, MboII, PleI, SapI and SfaNI, whereat BmpI is preferred.


[0043] The fragmentation of the synthesized cDNAs is such accomplished, that the most fragments possible are obtained in a desired range of size. Suitable techniques of fragmentation are familiar to the expert, whereat a random fragmentation is preferred. Particularly preferred is a fragmentation by means of sonication, whereat the expert can determine—by a series of tests preceding a preparative fragmentation and by variation of parameters like duration of acoustic irradiation and/or intensity within these tests—the respective conditions, which lead to a fragmentation in the desired range of size. Also suitable for fragmentation according to the invention are enzymatic digests with restriction enzymes recognizing 4-mer, 6-mer or 8-mer sequence elements.


[0044] The isolation of fragments in the desired range of length under removal of shorter or longer fragments can—if desired—be accomplished by means of any method, in which nucleic acid fragments are separated according to their length, preferably under non-denaturating conditions, e.g. by means of gel electrophoresis, capillary gel electrophoresis, selective precipitation with ammonium acetate/EtOH, whereat gel electrophoresis is preferred. Particularly preferred is agarose gel electrophoresis. During or after the termination of gel electrophoresis, the bands can e.g. be stained, excised and isolated from the gel by means of common methods.


[0045] In a preferred embodiment of the method according to the invention, the cDNA-fragments have a length in the range between 100 to 1000 bp, more preferably in the range between 200 to 600 bp.


[0046] The expert also knows about methods allowing to provide the isolated cDNA-fragments—the ends of which may have overhangs after the fragmentation—with blunt ends. This can be accomplished by means of common enzymatic methods, whereat however only those strategies are suitable for the method according to the invention, which do not alter the poly(dA)/(dT)-end of the fragments connected to the first binding partner. A suitable method e.g. is the treatment with Pwo-DNA-Polymerase or Pwo-DNA-Polymerase. In dependence on the fragmentation method used, the blunt-end-formation may also be abandoned. If one e.g. uses enzymatic fragmentation, blunt-end-formation is not necessary, since the adaptor of step f) can also be ligated “strictly”.


[0047] In the method according to the invention, the second binding partner is preferably bound to a solid carrier, e.g. a microscopic slide, a microtiter plate, pearls consisting of organic (e.g. agarose or polyacrylamide) or inorganic compounds, etc, whereat paramagnetic pearls are preferred. The expert is also familiar with methods for coupling the second binding partner to the solid carrier. The expert furthermore is familiar with conditions—according to the binding pair chosen—which allow to bind the cDNA-fragments to the second binding partner via their first binding partner and to remove the non-bound fragments from the mixture.


[0048] Suitable double-stranded adaptor molecules and methods for blunt-end-ligation of the blunt ends of the adaptor molecules to the blunt ends of the cDNA-fragments are known to the expert. The adaptor molecule comprises a recognition site for a restriction enzyme, whereat this restriction enzyme preferably is a restriction enzyme producing overhanging ends, and the recognition site of which extends to at least 6 bp, preferably 8 bp, in order to exclude or largely decrease the probability, that an undesired cleavage within the cDNA-fragments will occur at a later stage of the method according to the invention. Preferred restriction enzymes are AscI, NotI, SrfI, PacI, PmeI, SwaI as well as further restriction enzymes known to the expert, which recognize a 6-mer or 8-mer sequence. The cDNA-fragments bound to the solid carrier are preferably used to perform an in vitro-amplification in order to obtain sufficient amounts of material for the following process steps. Suitable in vitro-amplification methods are well known to the expert, whereat PCR is one preferred amplification method. The primer sequences, which are largely complementary to a strand of the adaptor molecule or to a sequence of the primer of step (a) or to a sequence complementary thereto, have anyhow to be chosen that way, that one primer is complementary to the end of the extended synthesis product of the other primer, thereby permitting exponential amplification.


[0049] In a particularly preferred embodiment of the method according to the invention, the cleavage by means of a restriction enzyme in step (h) is such accomplished, that the obtained fragment provides a single-stranded end with at least 2 adenine residues and whereat the adaptor molecule in step (i) preferably provides an overhanging end with the respective dT-residues complementary thereto. A fragment with two overhanging adenine residues is e.g. obtained by a digest with BmpI.


[0050] At the end of the method according to the invention, the ligation—preferably a directed ligation —of the cDNA-fragments into a suitable vector is accomplished. Examples of such vectors are known to the expert. Preferably, the aforementioned DNA-fragments are cloned into an expression vector. In case of an expression vector for E. coli, these are e.g. exemplified by pGEMEX, derivatives of pUC (e.g. pUC8), pBR322, pBlueScript, pGEX-2T, pET3b and pQE-8. Expression vectors in yeast are exemplified by pY100 and Ycpad1, whereas e.g. pKCR, PEFBOS, cDM8 and pCEV4 are to be mentioned for the expression in animal cells. Particularly suitable for the expression in insect cells is the baculovirus-expression vector pAcSGHisNT-A. If one desires no expression, it is also possible to clone into the vectors in a manner contrary to the frame. Within the vectors, the DNA-fragments are functionally linked to regulatory elements, which permit their expression in prokaryotic or eukaryotic host cells. Such vectors besides the regulatory elements (e.g. a promoter) also typically contain an origin of replication and specific genes, which allow the transformed host cells to be selected in dependence on a phenotype. Examples of regulatory elements for the expression in pro-karyotes, e.g. in E. coli, are the lac-, trp- or T7-promotor; respective regulatory elements for the expression in eukaryotes are the AOX1-promotor or the GAL1-promotor for yeast and the CMV-, SV40, RVS-40-promotor, CMV-enhancer or SV40-enhancer for the expression in animal cells. Further examples of suitable promoters are the metallothionein I—and the polyhedrin-promoter. In particular the expression vectors based on T7 are among the suitable vectors for the expression in bacteria (Rosenberg et al., Gene 56 (1987), 125) as well as pMSXND for the expression in mammalian cells (Lee and Nathans, J. Biol. Chem. 263 (1988), 3521). The introduction of the aforementioned vectors into the host cell can be accomplished by means of common methods, e.g. by means of calcium phosphate transfection, DEAE-Dextran-mediated transfection, by means of a transfection mediated by cationic lipids, by means of electroporation, infection, gene gun, etc. These methods are described in standard works of molecular biology.


[0051] The present invention also relates to a gene library that can be obtained by the method according to the invention. In a preferred embodiment, the gene library according to the invention is characterized in that at least one sequence of a gene or of a part of a gene is present, which encodes for a protein, which takes part in one of the following processes: amino acid synthesis, cellular metabolism, energy metabolism, fatty acid- and phospholipid-metabolism, purine-, pyrimidine-, nucleoside- and nucleotide-synthesis and —degradation, DNA-replication, transcription, translation, protein transport or protein binding.


[0052] In a more preferred embodiment the gene library according to the invention is characterized in that sequences of at least 50 genes or parts thereof, preferably of at least 500 genes or parts thereof and more preferred of at least 800 genes or parts thereof are present, the products of which are involved in the same or various of the afore mentioned processes.


[0053] The gene library according to the invention is characterized in that at least 95% of the sequences of the present genes have a length between 200 and 600 base pairs. The expert can achieve such a composition of the gene library by means of suitable methods, e.g. by the isolation of fragments according to these sizes in the intermediate steps of the afore mentioned method, e.g. by agarose gel electrophoresis and elution of fragments in the range of 200 to 600 bp from the gel in accordance with standard protocols.


[0054] In a further preferred embodiment, the gene library according to the invention is characterized in that the genes are derived from mouse, rat, dog, human, pig, hamster or cow. Suitable sources for these genes are familiar to the expert.


[0055] In an even more preferred embodiment, the gene library according to the invention is characterized in that (a) at least 60%, preferably at least 80% and yet more preferred at least 90% of the sequences comprise genes or parts of genes, which are derived from the 3′-region of the mRNA or that (b) at least 60% of the genes or of the parts thereof, preferably at least 80% and yet more preferred at least 90% comprise sequences without a poly(A)-tail.


[0056] Yet more preferred is a gene library according to the invention, which is characterized in that the sequences of the genes or of the parts thereof are present in a prokaryotic plasmid. In respect to suitable plasmids, we refer to the preceding embodiments.


[0057] In an even more preferred embodiment, the gene library according to the invention is characterized in that sequences of at least 50 genes or of parts thereof, preferably of 200 genes or of parts thereof and most preferred of 500 genes or of parts thereof are present, which are selected from:


[0058] (a) the sequences of the SEQ ID-list “Replication”,


[0059] (b) the sequences of the SEQ ID-list “Transcription”,


[0060] (c) the sequences of the SEQ ID-list “Translation”,


[0061] (d) the sequences of the SEQ ID-list “Transport- and binding Proteins”, or


[0062] (e) combinations of at least two of the groups (a) to (d).


[0063] The distinct sequences are given in the sequence protocol, which comprises the SEQ ID No. 1 to SEQ ID No. 840 and is an integral part of the patent application.


[0064] The present invention also refers to transformants containing the previously described gene library. These transformants encompass bacteria, yeast, insect—and animal cells, preferably mammalian cells. Favorable hosts are the E. coli strains HB101, DH1, DH10B, x1776, JM101, JM109, BL21, XL1Blue and SG 13009, the yeast species Saccharomyces cerevisiae and the animal cells L, 3T3, FM3A, CHO, COS, Vero, HeLa and the insect cell line sf9. Methods for transforming these host cells, methods for phenotypical selection of transformants and for expressing the DNA-sequences comprised within the gene library under employment of the above described vectors are known in this special field.






[0065]
FIG. 1: Schematic depiction of the distinct steps for producing a cDNA gene library as described in the following example






[0066] The following example illustrates the invention.


EXAMPLE


Production of a Gene Library From the mRNA of Mouse Liver

[0067] (A) Preparation of mRNA


[0068] The RNA was isolated from homogenized mouse liver by using a kit for RNA-purification (Trizol, Life Technologies, Rockville, USA), followed by employing a kit for mRNA-isolation (Dynabeads mRNA Purification Kit, Dynal A. S., Oslo, Norway) in accordance with the manufacturer's recommendations. A total of about 2 μg of mRNA was used to construct the gene library.


[0069] (B) Synthesis of a Double-Stranded cDNA


[0070] The synthesis of the cDNA-first strand by means of the Gibco “cDNA synthesis system” (Fa. GibcoBRL Life Technologies GmbH, Karlsruhe; Cat.No. 18267-013) was accomplished in accordance with the manufacturer's information by using the permutated primer 5′-ATG ATG CTG GAG TTT TTT TTT TTT TTT TTT VN-3′, whereat V is A, C or G and N is A, C, G or T. The primer additionally carried a biotin residue at its 5′-end. The nucleotides underlined represent a BpmI-restriction site (type II restriction enzyme). After degradation with RNAse H, DNA-polymerase I was used for second strand-synthesis and ligase was used for connecting the open Okazaki-fragments according to common conditions. In the following, the generated double-stranded DNA was precipitated with ethanol, dried and resuspended in buffer.


[0071] (C) Fragmentation of the cDNA by Means of Sonication


[0072] The cDNA was treated with ultrasound for 1 minute with a pulse of 0.9 by using the Misonex 2020 System (Fa. Misonix, Farmingdale, N.Y., USA). The successful sonication was verified by gel electrophoresis of an aliquot. After positive control of the aliquots, the whole reaction mixture was separated by means of agarose gel electrophoresis and fragments in the size range corresponding to the insert length of 200 to 600 bp, which is desired for the gene library to be produced, were eluted from the agarose gel by means of a “QIAquick gel extraction kit (Qiagen).


[0073] (D) Providing the Eluted cDNA-Fragments With Blunt Ends


[0074] After the sonication, the isolated DNA-fragments also provide non-specific 5′- or 3′-overhanging ends. These ends were filled or cleaved with Pwo-DNA-polymerase (Hoffman LaRoche, Basel, Switzerland) in order to generate blunt ends. To this aim, 30 μl of DNA-solution were mixed with 8 μl of a dNTP-mixture (200 μM per dNTP), 2 μl distillated water, 4.5 μl 10×Pwo-buffer (with MgSO4) and 0.5 μl (5 E/μl) Pwo-DNA-polymerase, followed by an incubation at 70° C. for 30 minutes. The reaction was then stopped on ice.


[0075] (E) Coupling the 3′-Ends to Streptavidin-Coated Paramagnetic Pearls (“SA-beads”)


[0076] Via their biotinylated 5′-end (corresponding to the 3′-end of the mRNA), the fragments were bound by the non-biotinylated fragments (corresponding to internal or 5′-regions of the mRNA) by binding to paramagnetic pearls (Dynabeads M-280 Streptavidin; Fa. Dynal, Oslo, Norway) according to the manufacturer's information. By means of the following washing step, the remaining fragments (without biotinylation) were removed.


[0077] (F) Ligation of a Double-Stranded Adaptor-Oligonucleotide


[0078] In the following, a double-stranded adaptor-oligonucleotide with an internal AscI-restriction site and the following sequence of 5′-CTA ATA CGA CTC ACT ATA GGG CGC GCC AGC GTG GTC GCG GCC GAG GT-3′; 3′-CAG CGC CGG CTC CA-5′, was blunt-end ligated under standard conditions to the fragment end opposing the biotinylated end of the DNA-fragments, whereby the DNA-fragments are still bound to the pearls. In this adaptor, the underlined sequence corresponds to the T7-promotor sequence, the sequence GGCGCGCC printed in boldface corresponds to the 5′-restriction site of AscI, which is used for cloning into the plasmid vector, and the sequence ACCTCGGCCGCGAC corresponds to the complementary “auxiliary” oligonucleotide. The reaction was performed overnight at 16° C. in a volume of 20 μl. The reaction mix contained 1 μl of the coupled DNA, 2 μl of T4-DNA-ligase (40 E/μl; Roche) and 5 μl of the double-stranded adaptor-oligonucleotide (final concentration: 2.5 μM).


[0079] (G) Direct PCR-Amplification of the 3′-Ends at the “SA-Beads”


[0080] In order to obtain sufficient material for the following steps of the process, the fragments still being bound to the paramagnetic pearls and corresponding to the 3′-regions of the transcripts were amplified by means of PCR, whereat the following primers were used: 5′-PCR-primer (5′ SAPCR): 5′-CTA ATA CGA CTC ACT ATA GGG C-3′; 3′-primer (3′ SAPCR): biotin-5′-ATG ATG CTG GAG TTT TTT TTT TTT TTT T-3′. Thereby, the 5′-PCR primer lies on the ligated adapter, whereas the biotinylated 3′-PCR primer lies on the first strand synthesis-primer (including the BpmI-restriction site). First, the optimal number of PCR-cycles was determined for an aliquot of the reaction in order to later use this number of cycles for the preparative approach. Thereby, 10 μl of the ligation mixture were used as a template, 2 μl Pwo-DNA-polymerase (Roche), 1 μl of both amplification primers 5′ SAPCR (10 μM) and 3′ SAPCR (10 μM) each, and 10 μl of dNTP-solution (concentration per dNTP: 1 mM). The reaction was performed in a volume of 50 μl in a DNA Thermal Cycler (GeneAmp PCR Systems 9700; Perkin Elmer Applied Biosystems, Weiterstadt). After an initial step of denaturation (5 min, 75° C.; 30 sec, 94° C.), a total of 30 cycles with the following profile was performed: 10 seconds at 94° C., 30 seconds at 60° C. and 90 seconds at 72° C. In the following, the process terminates with a final step of elongation for 5 minutes at 72° C. After the PCR-reaction, the PCR-products were purified by means of the “Quiaquick PCR Purification Kit” (Qiagen).


[0081] (H) Digest of the PCR-Products with the Restriction Enzyme BpmI (type IIS Enzyme)


[0082] In the next step, a restriction digest was performed by using the restriction enzyme BmpI (NEB, Beverly, Mass., USA). Since a BpmI-restriction site has been incorporated during the first strand synthesis (see (A)) in direct neighborhood to the poly(T)-tail of the cDNA, the restriction digest at the one hand releases the cDNA-molecules from the “SA-Beads”, and at the other hand—and except for two T-residues—cuts off the poly(T)-tails of the PCR-products from those ends of the DNA-fragments corresponding to the 3′-ends of the transcripts. The “SA-Beads” still carrying the poly(T)-tails are then separated from the cDNA-fragments, which correspond to the 3′-ends of the mRNA, and which are now present in a free form in the solution. The fragments were again loaded onto a 1% agarose gel and fragments having a length between 200 bp and 600 bp were excised and eluted as described above.


[0083] (I) Ligation of a Double-Stranded Adaptor Molecule to the cDNA-Molecules Corresponding to the 3′-Ends of the mRNA


[0084] In the next step, a 3′-TT-adaptor was ligated at the released fragment end corresponding to the 3′-end of the mRNA. This adapter provides an “open” XhoI-restriction site at its 3′-end. This adaptor had the following sequence: 5′-TCG AGC GGC CGC CCG GGC AGG TTT-3′; 3′-CG CCG GCG GGC CCG TCC A-5′. This adaptor in its left portion contains an open XhoI-restriction site and in its right portion a single-stranded overhanging end with TT, which is compatible to the 3′-overhanging AA of the cDNAs. The lower adaptor sequence is phosphorylated at its 5′-end. The reaction was performed overnight at 16° C. in a volume of 100 μl, whereat 30 μl of DNA-solution, 2 μl of T4 DNA-ligase (400 E/μl; Roche) and 20 μl of the double-stranded adaptor-DNA (210 μM) were used. When annealing the two complementary adaptor oligonucleotides, two overhanging TT-nucleotides are produced at one site—these nucleotides permitting a ligation to the BpmI-digested cDNA—whereas at the other site an open XhoI-restriction site is created, which is later used for the unidirectional cloning.


[0085] (J) Restriction Digest of the cDNA-Fragments with AscI


[0086] In the following, the AscI-restriction site of the ligated fragments obtained that way was opened by cleavage with AscI (NEB). The fragments thereafter carried an AscI-restriction site at their 5′-end and a XhoI-restriction site at their 3′-end. This was followed by another validation of the correct size by means of agarose gel electrophoresis, whereby fragments with the desired insert length (200 to 600 bp) were eluted from the gel as described above.


[0087] (K) Unidirectional Cloning


[0088] The eluted fragments were finally ligated in an orientated manner into a BlueScript derivative (pBSC-NTER; Fa. Stratagene, Heidelberg, Germany), which was such modified, that it contained within its polylinker the restriction sites compatible to the two restriction sites of the cDNA-fragments and thus allowed to be digested with the respective restriction enzymes. This was followed by a transformation into competent DH10B-cells (GibcoBRL Life Technologies GmbH, Karlsruhe). As a control, the cDNA-inserts were amplified with the PCR-primers “NPCR1” (5′-TCG AGC GGC CGC CCG GGC AGG T-3′) and “NPCR2” (5′-AGC GTG GTC GCG GCC GAG GT-3′), which are lying on the two adaptors. In this amplification, no additional parts of the vector were co-amplified.
1SEQ ID-List “Replication”SEQ ID No 25MOUSE: MG-13-4c19ReplicationSEQ ID No 26MOUSE: MG-13-4e3ReplicationSEQ ID No 46MOUSE: MG-14-4e9ReplicationSEQ ID No 53MOUSE: MG-15-1e3ReplicationSEQ ID No 65MOUSE: MG-16-3b14ReplicationSEQ ID No 69MOUSE: MG-16-4c5ReplicationSEQ ID No 79MOUSE: MG-16-6b20ReplicationSEQ ID No 85MOUSE: MG-16-7a13ReplicationSEQ ID No 92MOUSE: MG-16-7m2ReplicationSEQ ID No 98MOUSE: MG-16-9b6ReplicationSEQ ID No 100MOUSE: MG-16-9m14ReplicationSEQ ID No 104MOUSE: MG-19-2c13ReplicationSEQ ID No 112MOUSE: MG-3-100b4ReplicationSEQ ID No 118MOUSE: MG-3-106m10ReplicationSEQ ID No 119MOUSE: MG-3-106n16ReplicationSEQ ID No 122MOUSE: MG-3-108j10ReplicationSEQ ID No 123MOUSE: MG-3-108j3ReplicationSEQ ID No 160MOUSE: MG-3-12a9ReplicationSEQ ID No 161MOUSE: MG-3-12e6ReplicationSEQ ID No 169MOUSE: MG-3-13k7ReplicationSEQ ID No 174MOUSE: MG-3-142o11ReplicationSEQ ID No 182MOUSE: MG-3-15a14ReplicationSEQ ID No 183MOUSE: MG-3-15f4ReplicationSEQ ID No 187MOUSE: MG-3-18j5ReplicationSEQ ID No 190MOUSE: MG-3-1e22ReplicationSEQ ID No 203MOUSE: MG-3-22m14ReplicationSEQ ID No 209MOUSE: MG-3-23j6ReplicationSEQ ID No 216MOUSE: MG-3-25a15ReplicationSEQ ID No 217MOUSE: MG-3-25d11ReplicationSEQ ID No 220MOUSE: MG-3-25i17ReplicationSEQ ID No 221MOUSE: MG-3-25k21ReplicationSEQ ID No 225MOUSE: MG-3-26l10ReplicationSEQ ID No 241MOUSE: MG-3-2c8ReplicationSEQ ID No 244MOUSE: MG-3-2k6ReplicationSEQ ID No 250MOUSE: MG-3-31m18ReplicationSEQ ID No 268MOUSE: MG-3-37n1ReplicationSEQ ID No 276MOUSE: MG-3-3i15ReplicationSEQ ID No 277MOUSE: MG-3-3o3ReplicationSEQ ID No 282MOUSE: MG-3-43c4ReplicationSEQ ID No 285MOUSE: MG-3-43k7ReplicationSEQ ID No 290MOUSE: MG-3-44m16ReplicationSEQ ID No 291MOUSE: MG-3-45k11ReplicationSEQ ID No 297MOUSE: MG-3-46o2ReplicationSEQ ID No 300MOUSE: MG-3-47l23ReplicationSEQ ID No 310MOUSE: MG-3-49h15ReplicationSEQ ID No 313MOUSE: MG-3-4c14ReplicationSEQ ID No 354MOUSE: MG-3-64k13ReplicationSEQ ID No 367MOUSE: MG-3-71n17ReplicationSEQ ID No 379MOUSE: MG-3-75j6ReplicationSEQ ID No 380MOUSE: MG-3-75n23ReplicationSEQ ID No 382MOUSE: MG-3-76b11ReplicationSEQ ID No 399MOUSE: MG-3-7n12ReplicationSEQ ID No 401MOUSE: MG-3-80h21ReplicationSEQ ID No 402MOUSE: MG-3-80l14ReplicationSEQ ID No 405MOUSE: MG-3-81h2ReplicationSEQ ID No 415MOUSE: MG-3-85f14ReplicationSEQ ID No 420MOUSE: MG-3-88k7ReplicationSEQ ID No 425MOUSE: MG-3-8f8ReplicationSEQ ID No 438MOUSE: MG-3-95i1ReplicationSEQ ID No 439MOUSE: MG-3-95o9ReplicationSEQ ID No 441MOUSE: MG-3-96p21ReplicationSEQ ID No 450MOUSE: MG-4-145n21ReplicationSEQ ID No 452MOUSE: MG-4-145o24ReplicationSEQ ID No 454MOUSE: MG-4-146h14ReplicationSEQ ID No 462MOUSE: MG-4-148g1ReplicationSEQ ID No 492MOUSE: MG-6-13h17ReplicationSEQ ID No 496MOUSE: MG-6-15c24ReplicationSEQ ID No 519MOUSE: MG-6-24o2ReplicationSEQ ID No 521MOUSE: MG-6-29m3ReplicationSEQ ID No 531MOUSE: MG-6-31p16ReplicationSEQ ID No 535MOUSE: MG-6-3213ReplicationSEQ ID No 537MOUSE: MG-6-33j6ReplicationSEQ ID No 540MOUSE: MG-6-35k17ReplicationSEQ ID No 557MOUSE: MG-6-3i20ReplicationSEQ ID No 565MOUSE: MG-6-40e19ReplicationSEQ ID No 571MOUSE: MG-6-41g13ReplicationSEQ ID No 581MOUSE: MG-6-43p21ReplicationSEQ ID No 622MOUSE: MG-6-64m19ReplicationSEQ ID No 631MOUSE: MG-6-6p14ReplicationSEQ ID No 634MOUSE: MG-6-71j22ReplicationSEQ ID No 640MOUSE: MG-6-75a6ReplicationSEQ ID No 659MOUSE: MG-6-82p23ReplicationSEQ ID No 692MOUSE: MG-8-117o11ReplicationSEQ ID No 707MOUSE: MG-8-12n3ReplicationSEQ ID No 714MOUSE: MG-8-13n3ReplicationSEQ ID No 715MOUSE: MG-8-14g1ReplicationSEQ ID No 719MOUSE: MG-8-16a12ReplicationSEQ ID No 734MOUSE: MG-8-20o20ReplicationSEQ ID No 753MOUSE: MG-8-27k19ReplicationSEQ ID No 771MOUSE: MG-8-33b18ReplicationSEQ ID No 778MOUSE: MG-8-34d4ReplicationSEQ ID No 790MOUSE: MG-8-38p11ReplicationSEQ ID No 796MOUSE: MG-8-40g12ReplicationSEQ ID No 815MOUSE: MG-8-51c20ReplicationSEQ ID No 817MOUSE: MG-8-52d1ReplicationSEQ ID No 823MOUSE: MG-8-54g9Replication


[0089]

2











SEQ ID-List “Transcription”



















SEQ ID No 7
MOUSE: MG-11-1o21
Transcription



SEQ ID No 21
MOUSE: MG-13-1g21
Transcription



SEQ ID No 27
MOUSE: MG-13-4n3
Transcription



SEQ ID No 29
MOUSE: MG-13-6o14
Transcription



SEQ ID No 52
MOUSE: MG-14-5j23
Transcription



SEQ ID No 54
MOUSE: MG-15-2e20
Transcription



SEQ ID No 62
MOUSE: MG-16-10g4
Transcription



SEQ ID No 63
MOUSE: MG-16-10o16
Transcription



SEQ ID No 64
MOUSE: MG-16-2k3
Transcription



SEQ ID No 71
MOUSE: MG-16-5b5
Transcription



SEQ ID No 73
MOUSE: MG-16-5i18
Transcription



SEQ ID No 94
MOUSE: MG-16-8n16
Transcription



SEQ ID No 115
MOUSE: MG-3-102p7
Transcription



SEQ ID No 116
MOUSE: MG-3-103m15
Transcription



SEQ ID No 121
MOUSE: MG-3-108d19
Transcription



SEQ ID No 125
MOUSE: MG-3-109l17
Transcription



SEQ ID No 126
MOUSE: MG-3-109p13
Transcription



SEQ ID No 127
MOUSE: MG-3-10c1
Transcription



SEQ ID No 130
MOUSE: MG-3-10k3
Transcription



SEQ ID No 134
MOUSE: MG-3-110e4
Transcription



SEQ ID No 138
MOUSE: MG-3-113f21
Transcription



SEQ ID No 139
MOUSE: MG-3-113j5
Transcription



SEQ ID No 140
MOUSE: MG-3-113m21
Transcription



SEQ ID No 142
MOUSE: MG-3-114n3
Transcription



SEQ ID No 144
MOUSE: MG-3-116m5
Transcription



SEQ ID No 151
MOUSE: MG-3-11l11
Transcription



SEQ ID No 155
MOUSE: MG-3-122a21
Transcription



SEQ ID No 162
MOUSE: MG-3-12j14
Transcription



SEQ ID No 163
MOUSE: MG-3-12j20
Transcription



SEQ ID No 164
MOUSE: MG-3-12k8
Transcription



SEQ ID No 165
MOUSE: MG-3-136n16
Transcription



SEQ ID No 168
MOUSE: MG-3-13k15
Transcription



SEQ ID No 170
MOUSE: MG-3-140a7
Transcription



SEQ ID No 172
MOUSE: MG-3-140n23
Transcription



SEQ ID No 176
MOUSE: MG-3-143h24
Transcription



SEQ ID No 180
MOUSE: MG-3-14f24
Transcription



SEQ ID No 185
MOUSE: MG-3-18d4
Transcription



SEQ ID No 186
MOUSE: MG-3-18h18
Transcription



SEQ ID No 189
MOUSE: MG-3-19k16
Transcription



SEQ ID No 194
MOUSE: MG-3-20n20
Transcription



SEQ ID No 197
MOUSE: MG-3-21n21
Transcription



SEQ ID No 199
MOUSE: MG-3-22e2
Transcription



SEQ ID No 202
MOUSE: MG-3-22m1
Transcription



SEQ ID No 206
MOUSE: MG-3-23d22
Transcription



SEQ ID No 212
MOUSE: MG-3-24h16
Transcription



SEQ ID No 213
MOUSE: MG-3-24l17
Transcription



SEQ ID No 215
MOUSE: MG-3-24o2
Transcription



SEQ ID No 218
MOUSE: MG-3-25f1
Transcription



SEQ ID No 223
MOUSE: MG-3-26a2
Transcription



SEQ ID No 228
MOUSE: MG-3-27d20
Transcription



SEQ ID No 229
MOUSE: MG-3-27i13
Transcription



SEQ ID No 233
MOUSE: MG-3-28d3
Transcription



SEQ ID No 235
MOUSE: MG-3-28i9
Transcription



SEQ ID No 238
MOUSE: MG-3-29l11
Transcription



SEQ ID No 240
MOUSE: MG-3-29n9
Transcription



SEQ ID No 242
MOUSE: MG-3-2d4
Transcription



SEQ ID No 252
MOUSE: MG-3-32f14
Transcription



SEQ ID No 254
MOUSE: MG-3-32j16
Transcription



SEQ ID No 255
MOUSE: MG-3-32k3
Transcription



SEQ ID No 257
MOUSE: MG-3-33l21
Transcription



SEQ ID No 262
MOUSE: MG-3-36f22
Transcription



SEQ ID No 264
MOUSE: MG-3-37h11
Transcription



SEQ ID No 266
MOUSE: MG-3-37k2
Transcription



SEQ ID No 267
MOUSE: MG-3-37l19
Transcription



SEQ ID No 270
MOUSE: MG-3-38m21
Transcription



SEQ ID No 272
MOUSE: MG-3-38n16
Transcription



SEQ ID No 274
MOUSE: MG-3-39g12
Transcription



SEQ ID No 275
MOUSE: MG-3-3f9
Transcription



SEQ ID No 278
MOUSE: MG-3-40c9
Transcription



SEQ ID No 280
MOUSE: MG-3-41j5
Transcription



SEQ ID No 281
MOUSE: MG-3-41p22
Transcription



SEQ ID No 283
MOUSE: MG-3-43f6
Transcription



SEQ ID No 288
MOUSE: MG-3-44g1
Transcription



SEQ ID No 294
MOUSE: MG-3-45p20
Transcription



SEQ ID No 295
MOUSE: MG-3-46d2
Transcription



SEQ ID No 296
MOUSE: MG-3-46h17
Transcription



SEQ ID No 303
MOUSE: MG-3-48k13
Transcription



SEQ ID No 304
MOUSE: MG-3-48l10
Transcription



SEQ ID No 305
MOUSE: MG-3-48l24
Transcription



SEQ ID No 307
MOUSE: MG-3-48o8
Transcription



SEQ ID No 308
MOUSE: MG-3-49f13
Transcription



SEQ ID No 315
MOUSE: MG-3-4d16
Transcription



SEQ ID No 322
MOUSE: MG-3-4n9
Transcription



SEQ ID No 324
MOUSE: MG-3-50l9
Transcription



SEQ ID No 325
MOUSE: MG-3-50m3
Transcription



SEQ ID No 326
MOUSE: MG-3-50n19
Transcription



SEQ ID No 329
MOUSE: MG-3-51l17
Transcription



SEQ ID No 331
MOUSE: MG-3-52d14
Transcription



SEQ ID No 333
MOUSE: MG-3-52p2
Transcription



SEQ ID No 336
MOUSE: MG-3-55p16
Transcription



SEQ ID No 339
MOUSE: MG-3-57a6
Transcription



SEQ ID No 342
MOUSE: MG-3-5c4
Transcription



SEQ ID No 349
MOUSE: MG-3-62b3
Transcription



SEQ ID No 351
MOUSE: MG-3-63j4
Transcription



SEQ ID No 355
MOUSE: MG-3-66b13
Transcription



SEQ ID No 358
MOUSE: MG-3-66n16
Transcription



SEQ ID No 359
MOUSE: MG-3-66p18
Transcription



SEQ ID No 368
MOUSE: MG-3-72g16
Transcription



SEQ ID No 372
MOUSE: MG-3-74a16
Transcription



SEQ ID No 376
MOUSE: MG-3-74k7
Transcription



SEQ ID No 378
MOUSE: MG-3-75e7
Transcription



SEQ ID No 381
MOUSE: MG-3-75p4
Transcription



SEQ ID No 384
MOUSE: MG-3-76e9
Transcription



SEQ ID No 387
MOUSE: MG-3-77h22
Transcription



SEQ ID No 389
MOUSE: MG-3-78o12
Transcription



SEQ ID No 393
MOUSE: MG-3-79k7
Transcription



SEQ ID No 395
MOUSE: MG-3-7f17
Transcription



SEQ ID No 398
MOUSE: MG-3-7m20
Transcription



SEQ ID No 400
MOUSE: MG-3-7o16
Transcription



SEQ ID No 403
MOUSE: MG-3-81f11
Transcription



SEQ ID No 410
MOUSE: MG-3-84j15
Transcription



SEQ ID No 411
MOUSE: MG-3-84n2
Transcription



SEQ ID No 412
MOUSE: MG-3-85b13
Transcription



SEQ ID No 417
MOUSE: MG-3-87b16
Transcription



SEQ ID No 419
MOUSE: MG-3-88h12
Transcription



SEQ ID No 421
MOUSE: MG-3-88l7
Transcription



SEQ ID No 422
MOUSE: MG-3-88m17
Transcription



SEQ ID No 426
MOUSE: MG-3-8f9
Transcription



SEQ ID No 429
MOUSE: MG-3-90e16
Transcription



SEQ ID No 430
MOUSE: MG-3-90e5
Transcription



SEQ ID No 432
MOUSE: MG-3-91d6
Transcription



SEQ ID No 435
MOUSE: MG-3-93m16
Transcription



SEQ ID No 437
MOUSE: MG-3-94j23
Transcription



SEQ ID No 444
MOUSE: MG-3-9h15
Transcription



SEQ ID No 449
MOUSE: MG-4-145l6
Transcription



SEQ ID No 457
MOUSE: MG-4-146o24
Transcription



SEQ ID No 461
MOUSE: MG-4-148f6
Transcription



SEQ ID No 465
MOUSE: MG-4-149d20
Transcription



SEQ ID No 471
MOUSE: MG-4-2o2
Transcription



SEQ ID No 477
MOUSE: MG-4-4h6
Transcription



SEQ ID No 479
MOUSE: MG-4-5b17
Transcription



SEQ ID No 483
MOUSE: MG-4-5j9
Transcription



SEQ ID No 487
MOUSE: MG-6-10o24
Transcription



SEQ ID No 488
MOUSE: MG-6-10o9
Transcription



SEQ ID No 502
MOUSE: MG-6-16l16
Transcription



SEQ ID No 503
MOUSE: MG-6-16p8
Transcription



SEQ ID No 504
MOUSE: MG-6-17n24
Transcription



SEQ ID No 506
MOUSE: MG-6-19g10
Transcription



SEQ ID No 509
MOUSE: MG-6-1k5
Transcription



SEQ ID No 514
MOUSE: MG-6-22n21
Transcription



SEQ ID No 524
MOUSE: MG-6-2k8
Transcription



SEQ ID No 526
MOUSE: MG-6-30o15
Transcription



SEQ ID No 527
MOUSE: MG-6-31b11
Transcription



SEQ ID No 530
MOUSE: MG-6-31l15
Transcription



SEQ ID No 534
MOUSE: MG-6-32e14
Transcription



SEQ ID No 538
MOUSE: MG-6-33o17
Transcription



SEQ ID No 539
MOUSE: MG-6-35i22
Transcription



SEQ ID No 541
MOUSE: MG-6-36a15
Transcription



SEQ ID No 542
MOUSE: MG-6-36b13
Transcription



SEQ ID No 545
MOUSE: MG-6-36i7
Transcription



SEQ ID No 547
MOUSE: MG-6-37k24
Transcription



SEQ ID No 566
MOUSE: MG-6-40g17
Transcription



SEQ ID No 572
MOUSE: MG-6-41n2
Transcription



SEQ ID No 573
MOUSE: MG-6-41n6
Transcription



SEQ ID No 575
MOUSE: MG-6-41o6
Transcription



SEQ ID No 576
MOUSE: MG-6-42b15
Transcription



SEQ ID No 578
MOUSE: MG-6-42f2
Transcription



SEQ ID No 583
MOUSE: MG-6-44a22
Transcription



SEQ ID No 587
MOUSE: MG-6-46j8
Transcription



SEQ ID No 588
MOUSE: MG-6-47h20
Transcription



SEQ ID No 590
MOUSE: MG-6-47o6
Transcription



SEQ ID No 594
MOUSE: MG-6-48p1
Transcription



SEQ ID No 604
MOUSE: MG-6-56d22
Transcription



SEQ ID No 609
MOUSE: MG-6-57j16
Transcription



SEQ ID No 612
MOUSE: MG-6-59f3
Transcription



SEQ ID No 614
MOUSE: MG-6-61e6
Transcription



SEQ ID No 616
MOUSE: MG-6-61k11
Transcription



SEQ ID No 635
MOUSE: MG-6-71n14
Transcription



SEQ ID No 638
MOUSE: MG-6-74m9
Transcription



SEQ ID No 642
MOUSE: MG-6-75e5
Transcription



SEQ ID No 643
MOUSE: MG-6-75j14
Transcription



SEQ ID No 647
MOUSE: MG-6-78a4
Transcription



SEQ ID No 663
MOUSE: MG-6-86g1
Transcription



SEQ ID No 669
MOUSE: MG-6-89b13
Transcription



SEQ ID No 670
MOUSE: MG-6-89n5
Transcription



SEQ ID No 671
MOUSE: MG-6-90a24
Transcription



SEQ ID No 673
MOUSE: MG-6-90d18
Transcription



SEQ ID No 679
MOUSE: MG-6-92a3
Transcription



SEQ ID No 689
MOUSE: MG-8-117m12
Transcription



SEQ ID No 705
MOUSE: MG-8-12m9
Transcription



SEQ ID No 710
MOUSE: MG-8-13d9
Transcription



SEQ ID No 718
MOUSE: MG-8-15l7
Transcription



SEQ ID No 721
MOUSE: MG-8-16n13.
Transcription



SEQ ID No 723
MOUSE: MG-8-17i1
Transcription



SEQ ID No 728
MOUSE: MG-8-1e1
Transcription



SEQ ID No 731
MOUSE: MG-8-1i1
Transcription



SEQ ID No 736
MOUSE: MG-8-22d8
Transcription



SEQ ID No 740
MOUSE: MG-8-23c18
Transcription



SEQ ID No 741
MOUSE: MG-8-23d3
Transcription



SEQ ID No 743
MOUSE: MG-8-23m17
Transcription



SEQ ID No 744
MOUSE: MG-8-24g5
Transcription



SEQ ID No 751
MOUSE: MG-8-26o3
Transcription



SEQ ID No 768
MOUSE: MG-8-32e6
Transcription



SEQ ID No 775
MOUSE: MG-8-33f19
Transcription



SEQ ID No 785
MOUSE: MG-8-36n1
Transcription



SEQ ID No 788
MOUSE: MG-8-38i4
Transcription



SEQ ID No 793
MOUSE: MG-8-39p19
Transcription



SEQ ID No 800
MOUSE: MG-8-44m16
Transcription



SEQ ID No 801
MOUSE: MG-8-44m23
Transcription



SEQ ID No 804
MOUSE: MG-8-45c4
Transcription



SEQ ID No 809
MOUSE: MG-8-48e21
Transcription



SEQ ID No 810
MOUSE: MG-8-48i10
Transcription



SEQ ID No 813
MOUSE: MG-8-50d19
Transcription



SEQ ID No 816
MOUSE: MG-8-52c9
Transcription



SEQ ID No 818
MOUSE: MG-8-52i4
Transcription



SEQ ID No 820
MOUSE: MG-8-53d23
Transcription



SEQ ID No 825
MOUSE: MG-8-54i8
Transcription



SEQ ID No 826
MOUSE: MG-8-54o11
Transcription



SEQ ID No 827
MOUSE: MG-8-54p8
Transcription



SEQ ID No 828
MOUSE: MG-8-56h3
Transcription



SEQ ID No 838
MOUSE: MG-8-64i6
Transcription



SEQ ID No 839
MOUSE: MG-8-68a1
Transcription



SEQ ID No 840
MOUSE: MG-9-1i19
Transcription











[0090]

3











SEQ ID-List “Translation”



















SEQ ID No 6
MOUSE: MG-11-1k9
Translation



SEQ ID No 13
MOUSE: MG-12-1a19
Translation



SEQ ID No 15
MOUSE: MG-12-1e9
Translation



SEQ ID No 22
MOUSE: MG-13-1m9
Translation



SEQ ID No 36
MOUSE: MG-14-2n22
Translation



SEQ ID No 45
MOUSE: MG-14-4e8
Translation



SEQ ID No 50
MOUSE: MG-14-5f17
Translation



SEQ ID No 74
MOUSE: MG-16-5i4
Translation



SEQ ID No 75
MOUSE: MG-16-5j12
Translation



SEQ ID No 76
MOUSE: MG-16-5n6
Translation



SEQ ID No 81
MOUSE: MG-16-6d20
Translation



SEQ ID No 83
MOUSE: MG-16-6i6
Translation



SEQ ID No 89
MOUSE: MG-16-7h24
Translation



SEQ ID No 90
MOUSE: MG-16-7h7
Translation



SEQ ID No 91
MOUSE: MG-16-7j8
Translation



SEQ ID No 93
MOUSE: MG-16-7o11
Translation



SEQ ID No 97
MOUSE: MG-16-9b10
Translation



SEQ ID No 129
MOUSE: MG-3-10h13
Translation



SEQ ID No 131
MOUSE: MG-3-10k5
Translation



SEQ ID No 133
MOUSE: MG-3-10p9
Translation



SEQ ID No 153
MOUSE: MG-3-120g12
Translation



SEQ ID No 156
MOUSE: MG-3-122e22
Translation



SEQ ID No 166
MOUSE: MG-3-139d2
Translation



SEQ ID No 171
MOUSE: MG-3-140g13
Translation



SEQ ID No 175
MOUSE: MG-3-143d7
Translation



SEQ ID No 178
MOUSE: MG-3-14c24
Translation



SEQ ID No 179
MOUSE: MG-3-14d5
Translation



SEQ ID No 192
MOUSE: MG-3-1j20
Translation



SEQ ID No 196
MOUSE: MG-3-21i18
Translation



SEQ ID No 201
MOUSE: MG-3-22k6
Translation



SEQ ID No 204
MOUSE: MG-3-22n13
Translation



SEQ ID No 207
MOUSE: MG-3-23e1
Translation



SEQ ID No 208
MOUSE: MG-3-23j5
Translation



SEQ ID No 210
MOUSE: MG-3-23p7
Translation



SEQ ID No 219
MOUSE: MG-3-25f12
Translation



SEQ ID No 224
MOUSE: MG-3-26d7
Translation



SEQ ID No 226
MOUSE: MG-3-26l5
Translation



SEQ ID No 227
MOUSE: MG-3-26n4
Translation



SEQ ID No 236
MOUSE: MG-3-29a3
Translation



SEQ ID No 247
MOUSE: MG-3-30h20
Translation



SEQ ID No 249
MOUSE: MG-3-31c24
Translation



SEQ ID No 251
MOUSE: MG-3-32d21
Translation



SEQ ID No 269
MOUSE: MG-3-37o13
Translation



SEQ ID No 279
MOUSE: MG-3-41b21
Translation



SEQ ID No 309
MOUSE: MG-3-49g2
Translation



SEQ ID No 311
MOUSE: MG-3-49k8
Translation



SEQ ID No 312
MOUSE: MG-3-49o23
Translation



SEQ ID No 316
MOUSE: MG-3-4g14
Translation



SEQ ID No 317
MOUSE: MG-3-4g22
Translation



SEQ ID No 319
MOUSE: MG-3-4i2
Translation



SEQ ID No 321
MOUSE: MG-3-4m17
Translation



SEQ ID No 332
MOUSE: MG-3-52l2
Translation



SEQ ID No 334
MOUSE: MG-3-54f11
Translation



SEQ ID No 340
MOUSE: MG-3-5b3
Translation



SEQ ID No 341
MOUSE: MG-3-5b4
Translation



SEQ ID No 345
MOUSE: MG-3-5g23
Translation



SEQ ID No 346
MOUSE: MG-3-5k15
Translation



SEQ ID No 347
MOUSE: MG-3-61i4
Translation



SEQ ID No 352
MOUSE: MG-3-63k21
Translation



SEQ ID No 357
MOUSE: MG-3-66j9
Translation



SEQ ID No 366
MOUSE: MG-3-71g13
Translation



SEQ ID No 371
MOUSE: MG-3-73n22
Translation



SEQ ID No 373
MOUSE: MG-3-74a2
Translation



SEQ ID No 377
MOUSE: MG-3-75a4
Translation



SEQ ID No 383
MOUSE: MG-3-76b14
Translation



SEQ ID No 390
MOUSE: MG-3-79b21
Translation



SEQ ID No 392
MOUSE: MG-3-79j2
Translation



SEQ ID No 396
MOUSE: MG-3-7g1
Translation



SEQ ID No 397
MOUSE: MG-3-7l16
Translation



SEQ ID No 406
MOUSE: MG-3-81o13
Translation



SEQ ID No 414
MOUSE: MG-3-85e13
Translation



SEQ ID No 418
MOUSE: MG-3-88c2
Translation



SEQ ID No 433
MOUSE: MG-3-91j21
Translation



SEQ ID No 434
MOUSE: MG-3-92p15
Translation



SEQ ID No 436
MOUSE: MG-3-93m8
Translation



SEQ ID No 442
MOUSE: MG-3-9f22
Translation



SEQ ID No 446
MOUSE: MG-3-9o4
Translation



SEQ ID No 448
MOUSE: MG-4-145j12
Translation



SEQ ID No 451
MOUSE: MG-4-145o11
Translation



SEQ ID No 456
MOUSE: MG-4-146n10
Translation



SEQ ID No 464
MOUSE: MG-4-148n17
Translation



SEQ ID No 467
MOUSE: MG-4-1i7
Translation



SEQ ID No 468
MOUSE: MG-4-1k23
Translation



SEQ ID No 481
MOUSE: MG-4-5e23
Translation



SEQ ID No 485
MOUSE: MG-4-86c3
Translation



SEQ ID No 512
MOUSE: MG-6-21f24
Translation



SEQ ID No 515
MOUSE: MG-6-22n7
Translation



SEQ ID No 518
MOUSE: MG-6-24o16
Translation



SEQ ID No 523
MOUSE: MG-6-2j1
Translation



SEQ ID No 525
MOUSE: MG-6-2l11
Translation



SEQ ID No 533
MOUSE: MG-6-32e12
Translation



SEQ ID No 536
MOUSE: MG-6-33d5
Translation



SEQ ID No 551
MOUSE: MG-6-38p3
Translation



SEQ ID No 554
MOUSE: MG-6-3c6
Translation



SEQ ID No 562
MOUSE: MG-6-3o1
Translation



SEQ ID No 564
MOUSE: MG-6-3o16
Translation



SEQ ID No 570
MOUSE: MG-6-41g11
Translation



SEQ ID No 574
MOUSE: MG-6-41o19
Translation



SEQ ID No 582
MOUSE: MG-6-44a19
Translation



SEQ ID No 584
MOUSE: MG-6-44i2
Translation



SEQ ID No 601
MOUSE: MG-6-55g3
Translation



SEQ ID No 610
MOUSE: MG-6-58p10
Translation



SEQ ID No 620
MOUSE: MG-6-63c21
Translation



SEQ ID No 621
MOUSE: MG-6-64d16
Translation



SEQ ID No 623
MOUSE: MG-6-64p15
Translation



SEQ ID No 650
MOUSE: MG-6-80j23
Translation



SEQ ID No 657
MOUSE: MG-6-82l17
Translation



SEQ ID No 661
MOUSE: MG-6-83n11
Translation



SEQ ID No 667
MOUSE: MG-6-88k9
Translation



SEQ ID No 674
MOUSE: MG-6-90g23
Translation



SEQ ID No 680
MOUSE: MG-6-92a9
Translation



SEQ ID No 681
MOUSE: MG-8-10b11
Translation



SEQ ID No 683
MOUSE: MG-8-117a15
Translation



SEQ ID No 685
MOUSE: MG-8-117g16
Translation



SEQ ID No 686
MOUSE: MG-8-117i1
Translation



SEQ ID No 687
MOUSE: MG-8-117k3
Translation



SEQ ID No 690
MOUSE: MG-8-117m23
Translation



SEQ ID No 691
MOUSE: MG-8-117m3
Translation



SEQ ID No 694
MOUSE: MG-8-118j13
Translation



SEQ ID No 700
MOUSE: MG-8-11l12
Translation



SEQ ID No 713
MOUSE: MG-8-13m24
Translation



SEQ ID No 716
MOUSE: MG-8-14o16
Translation



SEQ ID No 729
MOUSE: MG-8-1e21
Translation



SEQ ID No 730
MOUSE: MG-8-1e23
Translation



SEQ ID No 732
MOUSE: MG-8-1i19
Translation



SEQ ID No 735
MOUSE: MG-8-21e1
Translation



SEQ ID No 738
MOUSE: MG-8-22p22
Translation



SEQ ID No 754
MOUSE: MG-8-27p12
Translation



SEQ ID No 756
MOUSE: MG-8-28g9
Translation



SEQ ID No 758
MOUSE: MG-8-29d18
Translation



SEQ ID No 766
MOUSE: MG-8-32a23
Translation



SEQ ID No 769
MOUSE: MG-8-32g19
Translation



SEQ ID No 772
MOUSE: MG-8-33c4
Translation



SEQ ID No 777
MOUSE: MG-8-34b8
Translation



SEQ ID No 779
MOUSE: MG-8-34p22
Translation



SEQ ID No 789
MOUSE: MG-8-38o15
Translation



SEQ ID No 795
MOUSE: MG-8-40e9
Translation



SEQ ID No 803
MOUSE: MG-8-44p6
Translation



SEQ ID No 805
MOUSE: MG-8-45n10
Translation



SEQ ID No 807
MOUSE: MG-8-47d16
Translation



SEQ ID No 814
MOUSE: MG-8-51a18
Translation



SEQ ID No 824
MOUSE: MG-8-54i18
Translation



SEQ ID No 832
MOUSE: MG-8-60f17
Translation











[0091]

4











SEQ ID-List “Transport-and Binding Proteins”

















SEQ ID No 1
MOUSE: MG-11-1a21
Transport and binding proteins


SEQ ID No 2
MOUSE: MG-11-1c3
Transport and binding proteins


SEQ ID No 3
MOUSE: MG-11-1e15
Transport and binding proteins


SEQ ID No 4
MOUSE: MG-11-1e9
Transport and binding proteins


SEQ ID No 5
MOUSE: MG-11-1g9
Transport and binding proteins


SEQ ID No 8
MOUSE: MG-11-1o3
Transport and binding proteins


SEQ ID No 9
MOUSE: MG-11-2d5
Transport and binding proteins


SEQ ID No 10
MOUSE: MG-11-2h21
Transport and binding proteins


SEQ ID No 11
MOUSE: MG-11-2n19
Transport and binding proteins


SEQ ID No 12
MOUSE: MG-11-2p7
Transport and binding proteins


SEQ ID No 14
MOUSE: MG-12-1c11
Transport and binding proteins


SEQ ID No 16
MOUSE: MG-12-1g3
Transport and binding proteins


SEQ ID No 17
MOUSE: MG-12-1g9
Transport and binding proteins


SEQ ID No 18
MOUSE: MG-12-1i15
Transport and binding proteins


SEQ ID No 19
MOUSE: MG-12-1k17
Transport and binding proteins


SEQ ID No 20
MOUSE: MG-13-1c17
Transport and binding proteins


SEQ ID No 23
MOUSE: MG-13-2a16
Transport and binding proteins


SEQ ID No 24
MOUSE: MG-13-2f1
Transport and binding proteins


SEQ ID No 28
MOUSE: MG-13-6j20
Transport and binding proteins


SEQ ID No 30
MOUSE: MG-13-6p18
Transport and binding proteins


SEQ ID No 31
MOUSE: MG-13-6p3
Transport and binding proteins


SEQ ID No 32
MOUSE: MG-14-1k21
Transport and binding proteins


SEQ ID No 33
MOUSE: MG-14-2c6
Transport and binding proteins


SEQ ID No 34
MOUSE: MG-14-2k21
Transport and binding proteins


SEQ ID No 35
MOUSE: MG-14-2n18
Transport and binding proteins


SEQ ID No 37
MOUSE: MG-14-2o15
Transport and binding proteins


SEQ ID No 38
MOUSE: MG-14-3b13
Transport and binding proteins


SEQ ID No 39
MOUSE: MG-14-3b19
Transport and binding proteins


SEQ ID No 40
MOUSE: MG-14-3j13
Transport and binding proteins


SEQ ID No 41
MOUSE: MG-14-3j17
Transport and binding proteins


SEQ ID No 42
MOUSE: MG-14-3k15
Transport and binding proteins


SEQ ID No 43
MOUSE: MG-14-4b13
Transport and binding proteins


SEQ ID No 44
MOUSE: MG-14-4c7
Transport and binding proteins


SEQ ID No 47
MOUSE: MG-14-5a20
Transport and binding proteins


SEQ ID No 48
MOUSE: MG-14-5d13
Transport and binding proteins


SEQ ID No 49
MOUSE: MG-14-5f16
Transport and binding proteins


SEQ ID No 51
MOUSE: MG-14-5h15
Transport and binding proteins


SEQ ID No 55
MOUSE: MG-15-2f14
Transport and binding proteins


SEQ ID No 56
MOUSE: MG-15-2j1
Transport and binding proteins


SEQ ID No 57
MOUSE: MG-15-2p22
Transport and binding proteins


SEQ ID No 58
MOUSE: MG-15-3a6
Transport and binding proteins


SEQ ID No 59
MOUSE: MG-15-3f12
Transport and binding proteins


SEQ ID No 60
MOUSE: MG-15-3k2
Transport and binding proteins


SEQ ID No 61
MOUSE: MG-15-3n13
Transport and binding proteins


SEQ ID No 66
MOUSE: MG-16-3f2
Transport and binding proteins


SEQ ID No 67
MOUSE: MG-16-3h10
Transport and binding proteins


SEQ ID No 68
MOUSE: MG-16-4a19
Transport and binding proteins


SEQ ID No 70
MOUSE: MG-16-4e24
Transport and binding proteins


SEQ ID No 72
MOUSE: MG-16-5g12
Transport and binding proteins


SEQ ID No 77
MOUSE: MG-16-5o12
Transport and binding proteins


SEQ ID No 78
MOUSE: MG-16-5p22
Transport and binding proteins


SEQ ID No 80
MOUSE: MG-16-6b4
Transport and binding proteins


SEQ ID No 82
MOUSE: MG-16-6f22
Transport and binding proteins


SEQ ID No 84
MOUSE: MG-16-6o13
Transport and binding proteins


SEQ ID No 86
MOUSE: MG-16-7d5
Transport and binding proteins


SEQ ID No 87
MOUSE: MG-16-7d8
Transport and binding proteins


SEQ ID No 88
MOUSE: MG-16-7g14
Transport and binding proteins


SEQ ID No 95
MOUSE: MG-16-9a14
Transport and binding proteins


SEQ ID No 96
MOUSE: MG-16-9a20
Transport and binding proteins


SEQ ID No 99
MOUSE: MG-16-9e1
Transport and binding proteins


SEQ ID No 101
MOUSE: MG-17-1a5
Transport and binding proteins


SEQ ID No 102
MOUSE: MG-19-1a15
Transport and binding proteins


SEQ ID No 103
MOUSE: MG-19-1a5
Transport and binding proteins


SEQ ID No 105
MOUSE: MG-19-2e7
Transport and binding proteins


SEQ ID No 106
MOUSE: MG-19-2i17
Transport and binding proteins


SEQ ID No 107
MOUSE: MG-19-2m13
Transport and binding proteins


SEQ ID No 108
MOUSE: MG-20-1i13
Transport and binding proteins


SEQ ID No 109
MOUSE: MG-20-1k23
Transport and binding proteins


SEQ ID No 110
MOUSE: MG-20-1k3
Transport and binding proteins


SEQ ID No 111
MOUSE: MG-20-1m3
Transport and binding proteins


SEQ ID No 113
MOUSE: MG-3-102c24
Transport and binding proteins


SEQ ID No 114
MOUSE: MG-3-102g18
Transport and binding proteins


SEQ ID No 117
MOUSE: MG-3-106b23
Transport and binding proteins


SEQ ID No 120
MOUSE: MG-3-107o14
Transport and binding proteins


SEQ ID No 124
MOUSE: MG-3-108l7
Transport and binding proteins


SEQ ID No 128
MOUSE: MG-3-10e15
Transport and binding proteins


SEQ ID No 132
MOUSE: MG-3-10m13
Transport and binding proteins


SEQ ID No 135
MOUSE: MG-3-110k6
Transport and binding proteins


SEQ ID No 136
MOUSE: MG-3-112g2
Transport and binding proteins


SEQ ID No 137
MOUSE: MG-3-113c13
Transport and binding proteins


SEQ ID No 141
MOUSE: MG-3-114h8
Transport and binding proteins


SEQ ID No 143
MOUSE: MG-3-116a13
Transport and binding proteins


SEQ ID No 145
MOUSE: MG-3-118i20
Transport and binding proteins


SEQ ID No 146
MOUSE: MG-3-119l11
Transport and binding proteins


SEQ ID No 147
MOUSE: MG-3-119n6
Transport and binding proteins


SEQ ID No 148
MOUSE: MG-3-11c14
Transport and binding proteins


SEQ ID No 149
MOUSE: MG-3-11h21
Transport and binding proteins


SEQ ID No 150
MOUSE: MG-3-11i23
Transport and binding proteins


SEQ ID No 152
MOUSE: MG-3-11m11
Transport and binding proteins


SEQ ID No 154
MOUSE: MG-3-121b13
Transport and binding proteins


SEQ ID No 157
MOUSE: MG-3-122j2
Transport and binding proteins


SEQ ID No 158
MOUSE: MG-3-123g9
Transport and binding proteins


SEQ ID No 159
MOUSE: MG-3-12a20
Transport and binding proteins


SEQ ID No 167
MOUSE: MG-3-139o21
Transport and binding proteins


SEQ ID No 173
MOUSE: MG-3-141p23
Transport and binding proteins


SEQ ID No 177
MOUSE: MG-3-14b17
Transport and binding proteins


SEQ ID No 181
MOUSE: MG-3-14h14
Transport and binding proteins


SEQ ID No 184
MOUSE: MG-3-16d22
Transport and binding proteins


SEQ ID No 188
MOUSE: MG-3-18o12
Transport and binding proteins


SEQ ID No 191
MOUSE: MG-3-1h10
Transport and binding proteins


SEQ ID No 193
MOUSE: MG-3-20a17
Transport and binding proteins


SEQ ID No 195
MOUSE: MG-3-20o24
Transport and binding proteins


SEQ ID No 198
MOUSE: MG-3-21o12
Transport and binding proteins


SEQ ID No 200
MOUSE: MG-3-22j14
Transport and binding proteins


SEQ ID No 205
MOUSE: MG-3-22o14
Transport and binding proteins


SEQ ID No 211
MOUSE: MG-3-24h15
Transport and binding proteins


SEQ ID No 214
MOUSE: MG-3-24o15
Transport and binding proteins


SEQ ID No 222
MOUSE: MG-3-25p22
Transport and binding proteins


SEQ ID No 230
MOUSE: MG-3-27i21
Transport and binding proteins


SEQ ID No 231
MOUSE: MG-3-27m22
Transport and binding proteins


SEQ ID No 232
MOUSE: MG-3-28a21
Transport and binding proteins


SEQ ID No 234
MOUSE: MG-3-28g4
Transport and binding proteins


SEQ ID No 237
MOUSE: MG-3-29e3
Transport and binding proteins


SEQ ID No 239
MOUSE: MG-3-29m14
Transport and binding proteins


SEQ ID No 243
MOUSE: MG-3-2j13
Transport and binding proteins


SEQ ID No 245
MOUSE: MG-3-2m2
Transport and binding proteins


SEQ ID No 246
MOUSE: MG-3-30c21
Transport and binding proteins


SEQ ID No 248
MOUSE: MG-3-30m22
Transport and binding proteins


SEQ ID No 253
MOUSE: MG-3-32h9
Transport and binding proteins


SEQ ID No 256
MOUSE: MG-3-32p8
Transport and binding proteins


SEQ ID No 258
MOUSE: MG-3-34e1
Transport and binding proteins


SEQ ID No 259
MOUSE: MG-3-34p17
Transport and binding proteins


SEQ ID No 260
MOUSE: MG-3-35b15
Transport and binding proteins


SEQ ID No 261
MOUSE: MG-3-35p13
Transport and binding proteins


SEQ ID No 263
MOUSE: MG-3-36h6
Transport and binding proteins


SEQ ID No 265
MOUSE: MG-3-37j5
Transport and binding proteins


SEQ ID No 271
MOUSE: MG-3-38m24
Transport and binding proteins


SEQ ID No 273
MOUSE: MG-3-39a16
Transport and binding proteins


SEQ ID No 284
MOUSE: MG-3-43i9
Transport and binding proteins


SEQ ID No 286
MOUSE: MG-3-44b1
Transport and binding proteins


SEQ ID No 287
MOUSE: MG-3-44f13
Transport and binding proteins


SEQ ID No 289
MOUSE: MG-3-44l17
Transport and binding proteins


SEQ ID No 292
MOUSE: MG-3-45n20
Transport and binding proteins


SEQ ID No 293
MOUSE: MG-3-45o3
Transport and binding proteins


SEQ ID No 298
MOUSE: MG-3-47c5
Transport and binding proteins


SEQ ID No 299
MOUSE: MG-3-47e5
Transport and binding proteins


SEQ ID No 301
MOUSE: MG-3-48a13
Transport and binding proteins


SEQ ID No 302
MOUSE: MG-3-48k1
Transport and binding proteins


SEQ ID No 306
MOUSE: MG-3-48o14
Transport and binding proteins


SEQ ID No 314
MOUSE: MG-3-4c20
Transport and binding proteins


SEQ ID No 318
MOUSE: MG-3-4i17
Transport and binding proteins


SEQ ID No 320
MOUSE: MG-3-4l8
Transport and binding proteins


SEQ ID No 323
MOUSE: MG-3-50k13
Transport and binding proteins


SEQ ID No 327
MOUSE: MG-3-51b24
Transport and binding proteins


SEQ ID No 328
MOUSE: MG-3-51g15
Transport and binding proteins


SEQ ID No 330
MOUSE: MG-3-51l8
Transport and binding proteins


SEQ ID No 335
MOUSE: MG-3-54h15
Transport and binding proteins


SEQ ID No 337
MOUSE: MG-3-56j8
Transport and binding proteins


SEQ ID No 338
MOUSE: MG-3-56n13
Transport and binding proteins


SEQ ID No 343
MOUSE: MG-3-5e21
Transport and binding proteins


SEQ ID No 344
MOUSE: MG-3-5g1
Transport and binding proteins


SEQ ID No 348
MOUSE: MG-3-61p4
Transport and binding proteins


SEQ ID No 350
MOUSE: MG-3-62j3
Transport and binding proteins


SEQ ID No 353
MOUSE: MG-3-63p1
Transport and binding proteins


SEQ ID No 356
MOUSE: MG-3-66i9
Transport and binding proteins


SEQ ID No 360
MOUSE: MG-3-68i14
Transport and binding proteins


SEQ ID No 361
MOUSE: MG-3-69a7
Transport and binding proteins


SEQ ID No 362
MOUSE: MG-3-69n2
Transport and binding proteins


SEQ ID No 363
MOUSE: MG-3-6b5
Transport and binding proteins


SEQ ID No 364
MOUSE: MG-3-70g5
Transport and binding proteins


SEQ ID No 365
MOUSE: MG-3-70n24
Transport and binding proteins


SEQ ID No 369
MOUSE: MG-3-73b19
Transport and binding proteins


SEQ ID No 370
MOUSE: MG-3-73f21
Transport and binding proteins


SEQ ID No 374
MOUSE: MG-3-74d18
Transport and binding proteins


SEQ ID No 375
MOUSE: MG-3-74f18
Transport and binding proteins


SEQ ID No 385
MOUSE: MG-3-76f2
Transport and binding proteins


SEQ ID No 386
MOUSE: MG-3-76n17
Transport and binding proteins


SEQ ID No 388
MOUSE: MG-3-77l18
Transport and binding proteins


SEQ ID No 391
MOUSE: MG-3-79e7
Transport and binding proteins


SEQ ID No 394
MOUSE: MG-3-79n2
Transport and binding proteins


SEQ ID No 404
MOUSE: MG-3-81g17
Transport and binding proteins


SEQ ID No 407
MOUSE: MG-3-82c19
Transport and binding proteins


SEQ ID No 408
MOUSE: MG-3-82l18
Transport and binding proteins


SEQ ID No 409
MOUSE: MG-3-82l20
Transport and binding proteins


SEQ ID No 413
MOUSE: MG-3-85c3
Transport and binding proteins


SEQ ID No 416
MOUSE: MG-3-86a7
Transport and binding proteins


SEQ ID No 423
MOUSE: MG-3-89m14
Transport and binding proteins


SEQ ID No 424
MOUSE: MG-3-8f7
Transport and binding proteins


SEQ ID No 427
MOUSE: MG-3-8l23
Transport and binding proteins


SEQ ID No 428
MOUSE: MG-3-90d1
Transport and binding proteins


SEQ ID No 431
MOUSE: MG-3-91 a10
Transport and binding proteins


SEQ ID No 440
MOUSE: MG-3-96e8
Transport and binding proteins


SEQ ID No 443
MOUSE: MG-3-9g4
Transport and binding proteins


SEQ ID No 445
MOUSE: MG-3-9h2
Transport and binding proteins


SEQ ID No 447
MOUSE: MG-4-145f3
Transport and binding proteins


SEQ ID No 453
MOUSE: MG-4-146f15
Transport and binding proteins


SEQ ID No 455
MOUSE: MG-4-146l4
Transport and binding proteins


SEQ ID No 458
MOUSE: MG-4-147d22
Transport and binding proteins


SEQ ID No 459
MOUSE: MG-4-148b6
Transport and binding proteins


SEQ ID No 460
MOUSE: MG-4-148b7
Transport and binding proteins


SEQ ID No 463
MOUSE: MG-4-148k6
Transport and binding proteins


SEQ ID No 466
MOUSE: MG-4-149e2
Transport and binding proteins


SEQ ID No 469
MOUSE: MG-4-2j8
Transport and binding proteins


SEQ ID No 470
MOUSE: MG-4-2o11
Transport and binding proteins


SEQ ID No 472
MOUSE: MG-4-3d20
Transport and binding proteins


SEQ ID No 473
MOUSE: MG-4-3f17
Transport and binding proteins


SEQ ID No 474
MOUSE: MG-4-3l13
Transport and binding proteins


SEQ ID No 475
MOUSE: MG-4-4g14
Transport and binding proteins


SEQ ID No 476
MOUSE: MG-4-4h1
Transport and binding proteins


SEQ ID No 478
MOUSE: MG-4-4i10
Transport and binding proteins


SEQ ID No 480
MOUSE: MG-4-5c24
Transport and binding proteins


SEQ ID No 482
MOUSE: MG-4-5i13
Transport and binding proteins


SEQ ID No 484
MOUSE: MG-4-6d16
Transport and binding proteins


SEQ ID No 486
MOUSE: MG-6-10g10
Transport and binding proteins


SEQ ID No 489
MOUSE: MG-6-11a6
Transport and binding proteins


SEQ ID No 490
MOUSE: MG-6-12c20
Transport and binding proteins


SEQ ID No 491
MOUSE: MG-6-13g8
Transport and binding proteins


SEQ ID No 493
MOUSE: MG-6-13k5
Transport and binding proteins


SEQ ID No 494
MOUSE: MG-6-14h9
Transport and binding proteins


SEQ ID No 495
MOUSE: MG-6-14k2
Transport and binding proteins


SEQ ID No 497
MOUSE: MG-6-15f10
Transport and binding proteins


SEQ ID No 498
MOUSE: MG-6-15m21
Transport and binding proteins


SEQ ID No 499
MOUSE: MG-6-15n12
Transport and binding proteins


SEQ ID No 500
MOUSE: MG-6-16c3
Transport and binding proteins


SEQ ID No 501
MOUSE: MG-6-16i3
Transport and binding proteins


SEQ ID No 505
MOUSE: MG-6-18c1
Transport and binding proteins


SEQ ID No 507
MOUSE: MG-6-19l15
Transport and binding proteins


SEQ ID No 508
MOUSE: MG-6-1g5
Transport and binding proteins


SEQ ID No 510
MOUSE: MG-6-1m13
Transport and binding proteins


SEQ ID No 511
MOUSE: MG-6-21e9
Transport and binding proteins


SEQ ID No 513
MOUSE: MG-6-22g14
Transport and binding proteins


SEQ ID No 516
MOUSE: MG-6-23i7
Transport and binding proteins


SEQ ID No 517
MOUSE: MG-6-24b11
Transport and binding proteins


SEQ ID No 520
MOUSE: MG-6-25i13
Transport and binding proteins


SEQ ID No 522
MOUSE: MG-6-2g18
Transport and binding proteins


SEQ ID No 528
MOUSE: MG-6-31b6
Transport and binding proteins


SEQ ID No 529
MOUSE: MG-6-31c20
Transport and binding proteins


SEQ ID No 532
MOUSE: MG-6-32c7
Transport and binding proteins


SEQ ID No 543
MOUSE: MG-6-36e10
Transport and binding proteins


SEQ ID No 544
MOUSE: MG-6-36h20
Transport and binding proteins


SEQ ID No 546
MOUSE: MG-6-36m12
Transport and binding proteins


SEQ ID No 548
MOUSE: MG-6-37m17
Transport and binding proteins


SEQ ID No 549
MOUSE: MG-6-38n21
Transport and binding proteins


SEQ ID No 550
MOUSE: MG-6-38n22
Transport and binding proteins


SEQ ID No 552
MOUSE: MG-6-39b7
Transport and binding proteins


SEQ ID No 553
MOUSE: MG-6-39o12
Transport and binding proteins


SEQ ID No 555
MOUSE: MG-6-3f17
Transport and binding proteins


SEQ ID No 556
MOUSE: MG-6-3h9
Transport and binding proteins


SEQ ID No 558
MOUSE: MG-6-3j21
Transport and binding proteins


SEQ ID No 559
MOUSE: MG-6-3k23
Transport and binding proteins


SEQ ID No 560
MOUSE: MG-6-3m16
Transport and binding proteins


SEQ ID No 561
MOUSE: MG-6-3n13
Transport and binding proteins


SEQ ID No 563
MOUSE: MG-6-3o12
Transport and binding proteins


SEQ ID No 567
MOUSE: MG-6-40h20
Transport and binding proteins


SEQ ID No 568
MOUSE: MG-6-40n24
Transport and binding proteins


SEQ ID No 569
MOUSE: MG-6-40p6
Transport and binding proteins


SEQ ID No 577
MOUSE: MG-6-42e21
Transport and binding proteins


SEQ ID No 579
MOUSE: MG-6-42j14
Transport and binding proteins


SEQ ID No 580
MOUSE: MG-6-42k8
Transport and binding proteins


SEQ ID No 585
MOUSE: MG-6-44k20
Transport and binding proteins


SEQ ID No 586
MOUSE: MG-6-45k11
Transport and binding proteins


SEQ ID No 589
MOUSE: MG-6-47k1
Transport and binding proteins


SEQ ID No 591
MOUSE: MG-6-48f16
Transport and binding proteins


SEQ ID No 592
MOUSE: MG-6-48n17
Transport and binding proteins


SEQ ID No 593
MOUSE: MG-6-48o4
Transport and binding proteins


SEQ ID No 595
MOUSE: MG-6-49c8
Transport and binding proteins


SEQ ID No 596
MOUSE: MG-6-49m7
Transport and binding proteins


SEQ ID No 597
MOUSE: MG-6-52n4
Transport and binding proteins


SEQ ID No 598
MOUSE: MG-6-53m5
Transport and binding proteins


SEQ ID No 599
MOUSE: MG-6-54d6
Transport and binding proteins


SEQ ID No 600
MOUSE: MG-6-55f18
Transport and binding proteins


SEQ ID No 602
MOUSE: MG-6-55j15
Transport and binding proteins


SEQ ID No 603
MOUSE: MG-6-55o21
Transport and binding proteins


SEQ ID No 605
MOUSE: MG-6-56e12
Transport and binding proteins


SEQ ID No 606
MOUSE: MG-6-57d20
Transport and binding proteins


SEQ ID No 607
MOUSE: MG-6-57g11
Transport and binding proteins


SEQ ID No 608
MOUSE: MG-6-57i2
Transport and binding proteins


SEQ ID No 611
MOUSE: MG-6-59c13
Transport and binding proteins


SEQ ID No 613
MOUSE: MG-6-61c23
Transport and binding proteins


SEQ ID No 615
MOUSE: MG-6-61h24
Transport and binding proteins


SEQ ID No 617
MOUSE: MG-6-61l13
Transport and binding proteins


SEQ ID No 618
MOUSE: MG-6-62e6
Transport and binding proteins


SEQ ID No 619
MOUSE: MG-6-62k22
Transport and binding proteins


SEQ ID No 624
MOUSE: MG-6-64p18
Transport and binding proteins


SEQ ID No 625
MOUSE: MG-6-65n22
Transport and binding proteins


SEQ ID No 626
MOUSE: MG-6-66l22
Transport and binding proteins


SEQ ID No 627
MOUSE: MG-6-68i24
Transport and binding proteins


SEQ ID No 628
MOUSE: MG-6-69f19
Transport and binding proteins


SEQ ID No 629
MOUSE: MG-6-69k15
Transport and binding proteins


SEQ ID No 630
MOUSE: MG-6-6n15
Transport and binding proteins


SEQ ID No 632
MOUSE: MG-6-70i21
Transport and binding proteins


SEQ ID No 633
MOUSE: MG-6-71g7
Transport and binding proteins


SEQ ID No 636
MOUSE: MG-6-71o8
Transport and binding proteins


SEQ ID No 637
MOUSE: MG-6-74c18
Transport and binding proteins


SEQ ID No 639
MOUSE: MG-6-75a14
Transport and binding proteins


SEQ ID No 641
MOUSE: MG-6-75c23
Transport and binding proteins


SEQ ID No 644
MOUSE: MG-6-76d14
Transport and binding proteins


SEQ ID No 645
MOUSE: MG-6-76f17
Transport and binding proteins


SEQ ID No 646
MOUSE: MG-6-77b14
Transport and binding proteins


SEQ ID No 648
MOUSE: MG-6-79i6
Transport and binding proteins


SEQ ID No 649
MOUSE: MG-6-79m7
Transport and binding proteins


SEQ ID No 651
MOUSE: MG-6-80k3
Transport and binding proteins


SEQ ID No 652
MOUSE: MG-6-81g14
Transport and binding proteins


SEQ ID No 653
MOUSE: MG-6-82c5
Transport and binding proteins


SEQ ID No 654
MOUSE: MG-6-82f1
Transport and binding proteins


SEQ ID No 655
MOUSE: MG-6-82h17
Transport and binding proteins


SEQ ID No 656
MOUSE: MG-6-82k5
Transport and binding proteins


SEQ ID No 658
MOUSE: MG-6-82m18
Transport and binding proteins


SEQ ID No 660
MOUSE: MG-6-83m8
Transport and binding proteins


SEQ ID No 662
MOUSE: MG-6-85j21
Transport and binding proteins


SEQ ID No 664
MOUSE: MG-6-86o11
Transport and binding proteins


SEQ ID No 665
MOUSE: MG-6-86p1
Transport and binding proteins


SEQ ID No 666
MOUSE: MG-6-86p2
Transport and binding proteins


SEQ ID No 668
MOUSE: MG-6-89a23
Transport and binding proteins


SEQ ID No 672
MOUSE: MG-6-90d16
Transport and binding proteins


SEQ ID No 675
MOUSE: MG-6-90m19
Transport and binding proteins


SEQ ID No 676
MOUSE: MG-6-91d19
Transport and binding proteins


SEQ ID No 677
MOUSE: MG-6-91l17
Transport and binding proteins


SEQ ID No 678
MOUSE: MG-6-91l19
Transport and binding proteins


SEQ ID No 682
MOUSE: MG-8-10l23
Transport and binding proteins


SEQ ID No 684
MOUSE: MG-8-117c22
Transport and binding proteins


SEQ ID No 688
MOUSE: MG-8-117l2
Transport and binding proteins


SEQ ID No 693
MOUSE: MG-8-118h19
Transport and binding proteins


SEQ ID No 695
MOUSE: MG-8-118k19
Transport and binding proteins


SEQ ID No 696
MOUSE: MG-8-11 b5
Transport and binding proteins


SEQ ID No 697
MOUSE: MG-8-11g1
Transport and binding proteins


SEQ ID No 698
MOUSE: MG-8-11g16
Transport and binding proteins


SEQ ID No 699
MOUSE: MG-8-11j13
Transport and binding proteins


SEQ ID No 701
MOUSE: MG-8-11n18
Transport and binding proteins


SEQ ID No 702
MOUSE: MG-8-12b4
Transport and binding proteins


SEQ ID No 703
MOUSE: MG-8-12i15
Transport and binding proteins


SEQ ID No 704
MOUSE: MG-8-12l22
Transport and binding proteins


SEQ ID No 706
MOUSE: MG-8-12n2
Transport and binding proteins


SEQ ID No 708
MOUSE: MG-8-12o8
Transport and binding proteins


SEQ ID No 709
MOUSE: MG-8-13a6
Transport and binding proteins


SEQ ID No 711
MOUSE: MG-8-13h22
Transport and binding proteins


SEQ ID No 712
MOUSE: MG-8-13i2
Transport and binding proteins


SEQ ID No 717
MOUSE: MG-8-15l22
Transport and binding proteins


SEQ ID No 720
MOUSE: MG-8-16f7
Transport and binding proteins


SEQ ID No 722
MOUSE: MG-8-17a6
Transport and binding proteins


SEQ ID No 724
MOUSE: MG-8-19a13
Transport and binding proteins


SEQ ID No 725
MOUSE: MG-8-19b7
Transport and binding proteins


SEQ ID No 726
MOUSE: MG-8-1a17
Transport and binding proteins


SEQ ID No 727
MOUSE: MG-8-1c19
Transport and binding proteins


SEQ ID No 733
MOUSE: MG-8-20h5
Transport and binding proteins


SEQ ID No 737
MOUSE: MG-8-22g2
Transport and binding proteins


SEQ ID No 739
MOUSE: MG-8-23a13
Transport and binding proteins


SEQ ID No 742
MOUSE: MG-8-23j1
Transport and binding proteins


SEQ ID No 745
MOUSE: MG-8-24h1
Transport and binding proteins


SEQ ID No 746
MOUSE: MG-8-25c24
Transport and binding proteins


SEQ ID No 747
MOUSE: MG-8-25c3
Transport and binding proteins


SEQ ID No 748
MOUSE: MG-8-25n7
Transport and binding proteins


SEQ ID No 749
MOUSE: MG-8-26g8
Transport and binding proteins


SEQ ID No 750
MOUSE: MG-8-26i19
Transport and binding proteins


SEQ ID No 752
MOUSE: MG-8-27j20
Transport and binding proteins


SEQ ID No 755
MOUSE: MG-8-28e17
Transport and binding proteins


SEQ ID No 757
MOUSE: MG-8-29d12
Transport and binding proteins


SEQ ID No 759
MOUSE: MG-8-29g17
Transport and binding proteins


SEQ ID No 760
MOUSE: MG-8-30c6
Transport and binding proteins


SEQ ID No 761
MOUSE: MG-8-31c6
Transport and binding proteins


SEQ ID No 762
MOUSE: MG-8-31e14
Transport and binding proteins


SEQ ID No 763
MOUSE: MG-8-31j16
Transport and binding proteins


SEQ ID No 764
MOUSE: MG-8-31o19
Transport and binding proteins


SEQ ID No 765
MOUSE: MG-8-31p21
Transport and binding proteins


SEQ ID No 767
MOUSE: MG-8-32c21
Transport and binding proteins


SEQ ID No 770
MOUSE: MG-8-32m4
Transport and binding proteins


SEQ ID No 773
MOUSE: MG-8-33e2
Transport and binding proteins


SEQ ID No 774
MOUSE: MG-8-33e20
Transport and binding proteins


SEQ ID No 776
MOUSE: MG-8-34a20
Transport and binding proteins


SEQ ID No 780
MOUSE: MG-8-35g12
Transport and binding proteins


SEQ ID No 781
MOUSE: MG-8-35n10
Transport and binding proteins


SEQ ID No 782
MOUSE: MG-8-36b7
Transport and binding proteins


SEQ ID No 783
MOUSE: MG-8-36g14
Transport and binding proteins


SEQ ID No 784
MOUSE: MG-8-36j7
Transport and binding proteins


SEQ ID No 786
MOUSE: MG-8-36n12
Transport and binding proteins


SEQ ID No 787
MOUSE: MG-8-38f12
Transport and binding proteins


SEQ ID No 791
MOUSE: MG-8-39h13
Transport and binding proteins


SEQ ID No 792
MOUSE: MG-8-39l1
Transport and binding proteins


SEQ ID No 794
MOUSE: MG-8-40b24
Transport and binding proteins


SEQ ID No 797
MOUSE: MG-8-40j12
Transport and binding proteins


SEQ ID No 798
MOUSE: MG-8-41d14
Transport and binding proteins


SEQ ID No 799
MOUSE: MG-8-42j10
Transport and binding proteins


SEQ ID No 802
MOUSE: MG-8-44p12
Transport and binding proteins


SEQ ID No 806
MOUSE: MG-8-46i18
Transport and binding proteins


SEQ ID No 808
MOUSE: MG-8-47m14
Transport and binding proteins


SEQ ID No 811
MOUSE: MG-8-49h21
Transport and binding proteins


SEQ ID No 812
MOUSE: MG-8-49l16
Transport and binding proteins


SEQ ID No 819
MOUSE: MG-8-52p7
Transport and binding proteins


SEQ ID No 821
MOUSE: MG-8-53g14
Transport and binding proteins


SEQ ID No 822
MOUSE: MG-8-53n12
Transport and binding proteins


SEQ ID No 829
MOUSE: MG-8-59b18
Transport and binding proteins


SEQ ID No 830
MOUSE: MG-8-59i18
Transport and binding proteins


SEQ ID No 831
MOUSE: MG-8-5h9
Transport and binding proteins


SEQ ID No 833
MOUSE: MG-8-60i3
Transport and binding proteins


SEQ ID No 834
MOUSE: MG-8-61n6
Transport and binding proteins


SEQ ID No 835
MOUSE: MG-8-62f20
Transport and binding proteins


SEQ ID No 836
MOUSE: MG-8-63o3
Transport and binding proteins


SEQ ID No 837
MOUSE: MG-8-63p15
Transport and binding proteins










[0092]


Claims
  • 1. Method for producing a gene library, whereat said method comprises the following steps: (a) Producing a double-stranded cDNA from a mRNA-population, wherein for the synthesis of the first cDNA-strand a primer is used, which at its 5′-end carries a first binding partner of a binding pair with this first binding partner having a binding affinity to a second binding partner of the binding pair, which at its 3′-end provides a poly(dT)-sequence being complementary to the poly(A)-tail of the mRNA, and—between the 5′-end being coupled to the first binding partner and the poly(dT)-sequence—a sequence, which as a double strand comprises a recognition site for a type II restriction enzyme; (b) Fragmentation of the cDNA obtained in step (a); (c) Binding the obtained fragments to the second binding partner, which is coupled to a solid carrier; (d) Ligating a double-stranded adaptor molecule to the fragment end opposing the end with the first binding partner, whereat the adaptor molecule contains a restriction site for a second restriction enzyme; (e) Performing an in vitro-amplification of the fragments bound to the carrier by means of a primer pair, whereat the first primer of the primer pair is mainly complementary to a strand of the adaptor molecule, and whereat the second primer of the primer pair at its 5′-end provides a first binding partner with an affinity to a second partner of a binding pair and has a sequence largely corresponding to the sequence of the primer of step (a) or a sequence complementary thereto; and (f) Ligating the obtained products into a vector.
  • 2. Method according to claim 1, wherein—after step (b)—the fragments having the desired length are isolated.
  • 3. Method according to claim 1 or 2, wherein—after step (b)— the obtained (isolated) fragments are provided with blunt ends.
  • 4. Method according to any of the claims 1 to 3, wherein the amplification products of step (e) are cleaved with a type II restriction enzyme, which recognizes the restriction site introduced in step (a).
  • 5. Method according to claim 4, wherein thereafter an adaptor molecule is ligated to the 5′-end of the PCR-products, which was obtained by the cleavage with the type II restriction enzyme.
  • 6. Method according to claim 5, wherein the products are cleaved with a second restriction enzyme, which recognizes the cleavage site introduced in step (d).
  • 7. Method according to claim 1, wherein the binding pair is biotin/avidin or biotin/streptavidin.
  • 8. Method according to claim 1, wherein the type II restriction enzyme is selected from the group consisting of BpmI, AlwI, BpsI, BpvI, Bci VI, BsaI, Bse RI, BsgI, Bsm AI, Earl, EciI, FokI, HgaI, HphI, MboII, PleI, SapI and SfaNI.
  • 9. Method according to any of the claims 1 to 8, wherein the primer for the first strand synthesis is a mixture of primers, which—at the 3′-side of the poly(dT)-sequence—provides the sequence 5′-VN-3′, wherein V is A, C or G and N is A, C, G or T.
  • 10. Method according to any of the claims 1 to 3, wherein the fragmentation in step (b) is a random fragmentation or an enzymatic fragmentation.
  • 11. Method according to claim 10, wherein the fragmentation is accomplished by sonication or an enzymatic (partial) digest.
  • 12. Method according to any of the claims 1 to 11, wherein the length of the fragments in step (b) is within a range of 200 to 600 bp.
  • 13. Method according to any of the claims 1 to 12, wherein the solid carrier in step (c) consists of paramagnetic pearls.
  • 14. Method according to any of the claims 1 to 13, wherein the restriction enzyme in step (d) is a restriction enzyme, which produces overhanging ends and/or which is different from the type II restriction enzyme of step (a).
  • 15. Method according to any of the claims 1 to 14, wherein the in vitro-amplification in step (e) is a PCR.
  • 16. Method according to claim 4 and 5, wherein the cleavage is such accomplished, that the obtained fragment provides an overhanging end with at least 2 adenine residues, whereat the adaptor molecule according to claim 5 provides an overhanging end complementary thereto.
  • 17. Gene library, which can be obtained by a method according to any of the claims 1 to 16.
  • 18. Gene library according to claim 17, wherein at least one sequence of a gene or a part of a gene is present, which codes for a protein being involved in one of the following processes selected from the group of: amino acid synthesis, cellular metabolism, energy metabolism, fatty acid- and phospholipid-metabolism, purine-, pyrimidine-, nucleoside- and nucleotide-synthesis and—degradation, DNA-replication, transcription, translation, protein transport or protein binding, characterized in that the gene library comprises at least 50 sequences, wherein at least 95% of the sequences of the presents genes or of their parts have a length between 200 and 600 base pairs.
  • 19. Gene library according to claim 17 or 18, wherein sequences of at least 200 genes or of parts thereof are present, the products of which are involved in the same or in various of the processes defined in claim 1.
  • 20. Gene library according to any of the claims 17 to 19, wherein sequences of at least 500 genes or of parts thereof are present, the products of which are involved in the same or in various of the processes defined in claim 1.
  • 21. Gene library according to any of the claims 17 to 20, wherein sequences of at least 800 genes or of parts thereof are present, the products of which are involved in the same or in various of the processes defined in claim 1.
  • 22. Gene library according to any of the claims 17 to 21, wherein the genes or the parts thereof are derived from the mouse, rat, dog, human, pig, hamster or cow.
  • 23. Gene library according to any of the claims 17 to 22, wherein at least 60% of the sequences comprise genes or parts of genes, which are derived from the 3′-region of the mRNA.
  • 24. Gene library according to any of the claims 17 to 23, wherein at least 60% of the genes or of the parts thereof comprise sequences without a poly(A)-tail.
  • 25. Gene library according to any of the claims 17 to 24, wherein the sequences of the genes or of the parts thereof are present in a prokaryotic plasmid.
  • 26. Gene library according to any of the claims 17 to 25, wherein the sequences are double-stranded.
  • 27. Gene library according to any of the claims 17 to 26, wherein sequences of at least 50 genes or of parts thereof are present, which are selected from: (a) the sequences of the SEQ ID-list “Replication”, (b) the sequences of the SEQ ID-list “Transcription”, (c) the sequences of the SEQ ID-list “Translation”, and (d) the sequences of the SEQ ID-list “Transport- and binding proteins”.
  • 28. Gene library according to claim 27, wherein sequences of at least 200 genes or of parts thereof are present, which are selected from: (a) the sequences of the SEQ ID-list “Replication”, (b) the sequences of the SEQ ID-list “Transcription”, (c) the sequences of the SEQ ID-list “Translation”, and (d) the sequences of the SEQ ID-list “Transport- and binding proteins”.
  • 29. Gene library according to claim 28, wherein sequences of at least 500 genes or of parts thereof are present, which are selected from: (a) the sequences of the SEQ ID-list “Replication”, (b) the sequences of the SEQ ID-list “Transcription”, (c) the sequences of the SEQ ID-list “Translation”, and (d) the sequences of the SEQ ID-list “Transport—and binding proteins”.
  • 30. Gene library according to any of the claims 27 to 29, wherein the sequences are selected from genes of at least two of the groups (a) to (d).
  • 31. Gene library according to any of the claims 17 to 30, wherein sequences of at least 50 genes or of parts thereof are present, which are selected from the SEQ ID No. 1 to SEQ ID No. 840.
  • 32. Gene library according to claim 31, wherein sequences of at least 200 genes or of parts thereof are present, which are selected from the SEQ ID No. 1 to SEQ ID No. 840.
  • 33. Gene library according to claim 31 or 32, wherein sequences of at least 500 genes or of parts thereof are present, which are selected from the SEQ ID No. 1 to SEQ ID No. 840.
  • 34. Transformant, which contains a gene library according to any of the claims 17 to 33.
Priority Claims (2)
Number Date Country Kind
201 03 510.3 Feb 2001 DE
01112165.4 May 2001 EP
PCT Information
Filing Document Filing Date Country Kind
PCT/EP02/01849 8/28/2003 WO