WO2001082688A1

WO2001082688A1 - Rab GDIα KNOCKOUT ANIMALS

Info

Publication number: WO2001082688A1
Application number: PCT/JP2000/008252
Authority: WO
Inventors: Science And Technology Corporation Japan
Original assignee: Takai, Yoshimi; Miyoshi, Jun; Ishizaki, Hiroyoshi; Okamoto, Miki
Priority date: 2000-04-28
Filing date: 2000-11-22
Publication date: 2001-11-08
Also published as: JP2001309734A

Abstract

Rab DGIα knockout animals which are a non-human individual, wherein a genomic gene encoding a low-molecular weight GTP-binding protein Rab GDIα has been substituted by a gene with the deletion of this function, and its offspring. These Rab DGIα knockout animals are useful in clarifying molecular mechanisms concerning memory and learning and developing diagnostic and therapeutic methods for neural diseases such as epilepsy, remedies therefor, etc. Moreover, these knockout animals are useful as model animals of neural disease XLMR.

Description

Specification

Rab GDI α; gene-deficient animal

The invention of this application relates to an animal lacking the Rab GDIa gene. More specifically, the invention of this application belong to the low molecular weight GTP-binding protein (G protein) Rab family one, Rab GDI _alpha expressed genes nerve tissue singular manner is substituted in the function-deficient mutant gene gene The present invention relates to a knockout animal that does not have the ability to synthesize Rab GDIa in the body or has a low ability to synthesize Rab GDIa, and a recombinant ES cell that is indispensable for producing this animal. This gene-deficient animal is useful in fields such as drug development for neurological diseases and the like. Background art

Neurotransmission is an extremely dynamic process involved in vesicle transport, association and fusion in the presynaptic plasma membrane. It plays a decisive role in synaptic plasticity, which is generally defined as a change in electrical activity dependent on the preceding stimulus (Reference 1). Short-term plasticity includes paired pulse facilitation (PPF), post-stimulation potentiation (PTP), and suppression, and duration ranges from tens of milliseconds to minutes (Ref. 2). PPF is a phenomenon in which the synaptic potential amplitude increases when synapses are activated by giving a pair of pulses at short intervals. The amount of response to the second stimulus usually increases depending on the interval between stimuli. PTP is a phenomenon in which the synaptic potential temporarily increases after a series of short stimuli. PTP is detected immediately after a series of stimuli, and decreases over the next few minutes. PPF and PTP result from increased probabilities of release of neurotransmitters from presynaptic nerve terminals triggered by increased ^{intraterminal} Ca ²⁺ , while repression of synapses after repeated actions is not complete, but not entirely. This is due to withdrawal of neurotransmitters that are easily released from the presynaptic nerve terminals. Synaptic suppression is a physiological defense mechanism against hyperexcitability, a potential cause of epileptic seizures. Long-term synaptic plasticity at excitatory synapses in the hippocampus includes long-term potentiation and suppression (LTP and LTD), and this plasticity is thought to be the basis of learning and memory (Ref. 3). Numerous biochemical processes Are known to be involved in vesicle release of neurotransmitters, but it remains unknown how these processes are involved in synaptic plasticity.

Rab GDI regulates the recycling of all low-molecular-weight G proteins of the Rab family involved in intracellular vesicle trafficking (References 4-10). Rab GDI exists from yeast to mammals. Mammalian Rab GDI constitutes a family of at least three members: Rab GDI a, one and one r, while yeast has the only gene essential for cell viability. Among the above three members, Rab GDIα is specifically expressed in neuronal tissues (Reference 11), and Rab GDI is expressed systemically. The tissue distribution of Rab GDI r has not yet been studied. Recent genetic analysis of X-linked non-specific mental retardation (XLMR) has shown that mutations in the Rab GDIa gene can cause this disease (Reference 12). Little is known about the gene defects associated with XLMR, but so far four mutations affecting FMR1, PAK3, Oligophrenin-1 and Rab GDIα have been reported to be responsible for XLMR. (Refs. 12–15). In particular, Rab GDIa is localized to the distal portion of chromosome Xq28 (Ref. 16), and mutations in the chromosome in this region have recently been reported to cause symptomatic diseases of XLMR, including epileptic seizures. (Ref. 17). These observations suggest that Rab GDI, which appears to be involved in the control of hyperexcitation, has a special function in vesicle trafficking of neuronal cells. Rab3A, a member of the Rab3 subfamily of Rab3A, 13B, 13C, and 3D, is a promising candidate protein that regulates neurotransmission through Rab GDIα (Reference 8). Studies on Rab3A-deficient mice in the CAI region of the hippocampus have yielded important findings on Rab3A function (Reference 18). Thus, in Rab3A-deficient neurons, synaptic depletion progresses rapidly, but the two forms of short-term synaptic plasticity (PPF and PTP) are not affected. These findings suggest that Rab3A may play a role in exocytosis. It is not known yet whether it will be. In order to elucidate the mechanisms of memory and learning, or the causes of neurological disorders such as epilepsy, it is necessary to analyze the synaptic function of two eurons at the molecular level. In order to understand such molecular mechanisms, it is indispensable to analyze the results of deletion of related genes at the individual level. The invention of this application has been made in view of the circumstances described above, and is an animal individual that is specifically expressed in nervous tissue and genetically deficient for Rab GDI α, which is a causative gene of XLMR for neuropsychiatric disorders. The challenge is to provide Disclosure of the invention

This application provides the following inventions (1) to (5) as inventions for solving the above-mentioned problems.

(1) Rab GDI a, a non-human animal individual that has generated totipotent cells in which the genomic gene encoding the low-molecular-weight GTP-binding protein Rab GDI α has been replaced by a mutant gene with a loss of function, and its descendants Gene-deficient animals.

(2) The Rab GDI o! Gene-deficient animal according to the invention (1), wherein the non-human animal is a mouse.

(3) The animal-derived tissue or cell according to the invention (1) or (2).

(4) ES cells in which the genomic gene encoding the low molecular weight GTP-binding protein Rab GDI has been replaced by a mutant gene lacking its function.

(5) The ES cell according to the invention (4), which is derived from a mouse. BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the strategy for gene targeting of the Rab GDIa gene. a: The top is the structure of the mouse Rab GDIa gene having 10 exons. Targeting vector is exon It was designed to remove a portion of 2 followed by exons 3-6. This construct contained a 4.4 kb 5 flanking sequence and a 4.8 kb 3 ′ flanking sequence. The MC1-neo cassette was inserted into the Sacl site inside exon 2, while the 5 'splice acceptor site remained intact. The diphtheria toxin DT-A cassette was inserted at the 3 'end. In the targeted allele, the MC1-Neo cassette replaces 2.4 kb of genomic DNA region. Homologous recombination was confirmed using restriction fragments and probes. b: Southern hybridization using Xbal digested DNA extracted from mouse tail and 3 ′ external probe shown in la. Mouse genotypes were identified by an 8.4 kb wild type (WT) and 5.5 kb mutant (MT) fragment. c: Genotyping by PCR analysis of DNA extracted from 21-day-old littermates. PCR primers were selected to produce a 366 bp DNA product representing the wild-type (WT) allele or a 224 bp product from the targeted (MT) Rab GDIa allele. d: ゥ stamp lot analysis of proteins extracted from brain of each genotype. A 60 kD band of Rab GDIa protein was detected. e: Western plot analysis of synaptic proteins extracted from the brains of wild-type and Rab GDIα-deficient mice.

FIG. 2 shows the results of testing the reduction of synaptic suppression during repeated stimulation in Rab GDIa-deficient mice. a: EPSC in response to repetitive stimulation (40 stimuli at 14 Hz) of the Scheffer minor axon Z commissure (SCC) tract was found to be wild-type (WT) in the presence of 50 Λ <D D—AP5. ) And Rab GDI-deficient mice (KO) were recorded in CA1 neurons in slices obtained. b: Amplitude of EPSC plotted as a function of number of stimuli during repeated stimuli in wild type (open circles, n = 13) and Rab GDIa deficient mice (solid circles, n = 16). EPSC amplitude was corrected for the amplitude of the first EPSC in the series of stimuli. Ten consecutive EPSCs were recorded every 30 seconds for each cell and averaged off-line. Differences between the two groups were significant at the time indicated by an asterisk (* P <0.05, t-test).

FIG. 3 shows the results of testing PPF enhancement in Rab GDI α-deficient mice. a: Wild-type (WT, upper trace) and Rab GDI or responsive to counterstimuli delivered to the SCC tract at various interstimulus intervals (50-300 ms) in the presence of 50 mm D-AP5. EPSCs recorded in CA1 neurons of deficient mice (KO, lower trace). b: PPF size. The ratio of the second versus first EPSC amplitude in wild-type (open circles, n = 17) and Rab GDIa-deficient mice (solid circles, n = 15) is plotted as a function of interstimulus interval. The PPF at 50 ms and 70 ms was different between the two groups (* P <0.05, t-test). FIG. 4 shows LTP at SCC-CA1 synapse in wild-type and Rab GDIa-deficient mice. LTP was induced by two 1-second twitches at 100 Hz applied 20 seconds apart. There is no significant difference in LTP measured at 60 minutes post-tetanus between wild type (open circles, n = 9) and Rab GDIα-deficient mice (solid circles, n = 7; P> 0.7, t-test) Was. Best mode for carrying out the invention.

In the Rab GDIa gene-deficient animal of the invention (1) provided by this application, the genomic gene encoding the protein Rab GDIα that is specifically expressed in nerve tissue is replaced with a mutant gene having a loss of function. It is a gene knockout non-human animal that has developed totipotent cells. More specifically, the Rab GDIα gene-deficient animal of the present invention (1) is provided as a heterozygote or a homozygote in which the Rab GDIa gene on the somatic chromosome is substituted with a mutant sequence thereof. The Rab GDIa gene-deficient animal of the present invention (1) can be prepared by a known target gene recombination method (gene targeting method: Science 244: 1288-1292, 1989). In this target gene recombination method, ES (embryonic stem) cells and the like are used as totipotent cells. ES cells are mouse (Nature 292: 154-156, 1981), rat (Dev. Biol. 163 (1): 288-292, 1994), Sazore (Proc. Natl. Acad. Sci. USA 92 (17): 7844-7848, 1995) and Egret (Mol. Reprod. Dev. 45 (4): 439-443, 1996). For pigs, EG (embryonic germ) cell strength has been established (Biol. Reprod 57 (5): 1089-1095, 1997). Therefore, the Rab GDIa gene-deficient animal of the present invention (1) can be produced for these animal species, and a mouse having a technique for producing a gene knockout animal is most suitable. The specific procedure of the production will be described below by taking the mouse of the invention (2) as an example. First, a genomic DNA fragment of the Rab GDI α gene is isolated, the DNA fragment is subjected to gene manipulation in a test tube, and the DNA fragment containing the initiation codon of the Rab GDI a gene is modified. Create a mutant DNA fragment that deletes the function of the a gene. Rab GDI a Geno For example, isolation of the mouse genomic DNA library can be obtained by screening a mouse genomic DNA library using an oligonucleotide probe synthesized based on a known human Rab GDIa cDNA sequence (GenBnk Accession No. D45021) or the like. . The target genomic DNA can also be obtained by PCR using a synthetic oligonucleotide corresponding to a part or both ends of the Rab GDI cDNA as a primer. In the case of targeting animals other than mice, the Rab GDIα gene of each animal is isolated by the above-mentioned method using the oligonucleotide synthesized based on the above cDNA sequence as a probe or primer. be able to. A targeting vector for modifying a part of the genomic DNA of the mouse Rab GDIa gene obtained by the method described above and introducing a mutation into the Rab GDIa gene of totipotent cells (ES cells) is known in the art. It is prepared according to a method (for example, Science 244: 1288-1292, 1989). For example, by replacing a part of the genomic DNA of the Rab GDI α gene with a cytotoxic resistance gene such as G418 (eg, neomycin resistance gene), or by replacing the cytotoxic resistance gene with the Rab GDIa gene genome By inserting a part of the DNA, a recombinant plasmid DNA having a mutant gene having a sequence homologous to the genomic DNA of Rab GDIa at both ends, that is, a targeting vector is prepared. In addition, a sequence such as a PGK1 promoter for controlling the expression and a PGK1 polyadenylation signal can be linked to the cytotoxic resistance gene. Further, the genomic DNA region of the Rab GDI or gene replaced or inserted by the cytotoxic resistance gene is preferably a genomic DNA region including an exon region including an initiation codon. The targeting vector for introducing a mutation into a part of the genomic DNA of the Rab GDI α gene is not particularly limited except that it has a sequence homologous to the genomic DNA of the Rab GDI α gene. Such sequences as drug resistance gene, cell selection gene (for example, diphtheria toxin A gene or herpes virus thymidine kinase gene), promoter, and enhancer can be used in an appropriate combination. Next, this targeting vector is introduced into mouse ES cells according to a known method. this As such a gene transfer method, a known electric pulse method, ribosome method, calcium phosphate method, or the like can be used, but in view of the efficiency of homologous gene recombination of the introduced gene, an electric pulse method for ES cells is preferable. The DNA of each transfected ES cell is extracted and the homologous gene set between the wild-type Rab GDI a gene present on the chromosome and the introduced Rab GDI a mutant gene fragment is analyzed by Southern blot analysis, PCR assay, etc. Cells that have undergone a recombination and have a mutation in the Rab GDIa gene on the chromosome are selected. The ES cells having the mutated gene thus obtained are injected into blastocysts of wild-type mice, and then the chimeric embryo is transplanted into the uterus of a foster mother. After the offspring are reared and reared, chimeric animals in which the Rab GDIa mutant gene has entered germline cells are selected. Selection is performed by extracting DNA from hair color differences or a part of the body (for example, the tip of the tail), and performing Southern blot analysis or PCR assay. Southern plot analysis and PCR using progeny obtained by crossing a wild-type animal with a chimeric animal in which a Rab GDI α mutant gene has entered a germline cell and using DNA extracted from a part of the body (for example, the end of the tail) as a material Perform Atsusei et al. To identify heterozygotes into which the Rab GDIα mutant gene has been introduced. The heterozygote containing the created Rab GDIa mutant gene stably carries the Rab GDI o; gene mutation in all of the cultured cells and somatic cells, and the offspring can efficiently transfer the mutation by mating etc. Can be transmitted to animals. Invention (3) is a tissue or cell isolated from the Rab GDIα gene-deficient animal of the invention (1) or (2). Specifically, it is a central nervous system tissue of an animal, such as a hippocampal slice or a neuron thereof. These tissues and cells can be used as a screening system for therapeutic agents for neurological diseases. Hereinafter, the present invention will be described in more detail and specifically with reference to Examples, but the present invention is not limited to the following Examples. Example

1. Method

1.1 DNA library screening

Rab GDI or cDNA was isolated from the mouse brain cDNA library A TriplEx (Clonetch) using bacterial strains and manufacturer's protocol and sequenced using the ABI DNA sequencer. A cDNA fragment encoding the N-terminal half region of Rab GDIa was subcloned into an appropriate plasmid vector and used as a probe for homology screening of the 129SVJ mouse genomic library λ FIXII (Stratagene). 1.2 Production of Rab GDI-deficient mice

A targeting vector was prepared by replacing exon 2 and the subsequent 3 'half of exons 3 to 6 with a neo-resistance gene cassette, transfected into RW4 ES cells, and followed the procedure described in reference 21. Selected. Homologous recombination was confirmed by Southern hybridization using 5'- and 3'_ external probes and a neo-resistance gene probe. Rab GDIa-deficient ES cells were microinjected into E3.5 C57BL / 6J blastula and transferred to MCH pseudopregnant surrogates to produce chimeras, which were crossed with BDF1 mice for germline transmission. Mice carrying the mutant allele were also backcrossed to C57BL / 6 mice. Genotyping was performed using the Rab GDI o in which the primers in the neo gene (SEQ ID NOs: 1 and 2) were replaced; the primers in the gene (SEQ ID NOs: 3 and 4) were used for Southern hybridization and PCR. Was performed. The PCR mixture was denatured at 95 ° C for 2 minutes and annealed at 55 ° C for 1 minute. The PCR was performed for 25 minutes at 72 ° C. for 2 minutes, at 95 ° C. for 30 seconds, and at 55 ° C. for 1 minute. The sample was extended at 72 ° C for an additional 5 minutes. PCR products were visualized on 4% NuSieve agarose (Takara) TAE (3: 1) gel. 1.3 Western plot analysis

An anti-Rab GDIa antibody was prepared against the C-terminal region of Rab GDIa fused to the GST protein, that is, 365 to 447 amino acid residues. Mouse brain is in lysis buffer of 320mM sucrose, 20mM Tris-C1 pH 7.5s 2mM EDTA and 10mM phenylmethylsulfonyl fluoride Was homogenized. Proteins of 50 «g were separated by SDS-PAGE, transferred to Immobilon membrane (Millipore), and blocked for 1 hour in Tris-buffered saline containing 5% of serum albumin. After 1 hour incubation with anti-Rab GDIa antibody and then 1 hour with peroxidase conjugated secondary antibody, blots were developed using ECL (Amersham Pharmacia Biotech).

1.4 Electrophysiological analysis

Transhippocampal slices (0.3-0.4 mm thick) were prepared from wild-type and mutant mice (4-10 weeks of age). The composition of the external recording solution: NaCl of 127 mM, 1.5 mM of KC and KH ₂ P0 ₄ of 1.2 mM, MgSO ₄ in 1.3 mM, and the NaHCO ₃ and lOmM of glucose of CaCl _2, 26 mM of 2.4 mM. To this solution 95% 0 ₂ and 5% C0 ₂ was saturated. Electrical stimulation (test pulse at 0.1 Hz, duration of 0.1 ms, intensity of about 200 // A) was performed from concentric bipolar electrodes inserted into the radial layer in the CA1 region. Extracellular and whole cell patch clamp recordings were performed at room temperature (24-26 ° C). In whole cell experiments, picrotoxin (100 μM) was added to block GABAergic inhibition. A cut was made between the CA1 and CA2 regions to block the transmission of burst activity from the CA2 / 3 region. A patch pipette contains an internal solution (pH 7.2) containing 150 mM cesium gluconate, 0.2 mM EGTA, 8 mM NaCl, 10 mM HEPES, 2 mM Mg ²⁺ ATP and 5 mM QX-314 (Sigma). Filled. Recordings were made using an axopateh ID amplifier (Axon Instruments). The signal was filtered at 2 kHz, digitized at 10 kHz and analyzed using pClainp software (Axon Instruments). EPSC amplitude or field EPSP initial slope was measured and these data were expressed as mean soil SEM as a percentage of the baseline. Statistical analysis used Student's t-test, with P <0.05 as the statistical significance level. 2. Result

2.1 Production of Rab GDI α-deficient mice

The mouse Rab GDIa gene consists of 10 exons spanning a 6.8 kb DNA region. Rab GDI α-deficient ES cells contain four exons (exon 3 to exon) of the Rab GDI α gene. Using a targeting vector designed to delete the 2.3 kb genomic DNA containing the DNA (up to 6), homologous recombination was performed (Fig. 1a). When the targeting vector was electroporated into RW4 ES cells, two G418-resistant colonies homozygous for the Rab GDIa gene were obtained because the Rab GDI gene is located on the X chromosome. The genotype of G418 resistant colonies was confirmed by Southern hybridization. Chimeric mice were produced using these ES clones. Since all Rab GDIa heterozygotes were female, male chimeric mice and female Rab GDIa heterozygotes were cross-bred to produce homozygous mutant progeny (FIGS. 1b and c).

Homozygous mice in which alleles were disrupted did not express any Rab GDI protein (Fig. 1d). Biochemical studies in Rab3A-deficient mice showed that labfilin 3 levels were selectively reduced to almost 30% of wild-type mice (Reference 18), whereas Rab GDIa-deficient mice had Rab3A, Rabll Other synaptic proteins, including rabphyrin 3, syntaxin, synabtotagmin I, synaptophysin and sinabsin I aZb, were normal (Fig. 1e). Rab GDIa-deficient mice were viable and fertile, and showed no morphological abnormalities in the brain, indicating that Rab GDI was not essential for nerve development.

2.2 Reduced synaptic depression during repeated stimulation in Rab GDI α-deficient mice

To investigate the physiological function of Rab GDI in controlling synaptic transmission in the central nervous system, it was investigated whether deletion of Rab GDIa alters synaptic transmission and activity-dependent plasticity at excitatory synapses in the CA1 region of the hippocampus. Tested. It has already been demonstrated that deletion of Rab3A in nerve endings accelerates synaptic suppression during repeated stimulation of the CA1 region (Reference 18). Therefore, changes in this form of presynaptic plasticity were tested. Repetitive stimulation at moderate frequencies (14 Hz) in wild-type mice elicited an initial enhancement of excitatory postsynaptic current (EPSC) amplitude, followed by a slight suppression (FIG. 2a, top trace). In contrast, the mutant synapse showed little or no suppression, and a large enhancement was accumulated during this continuous stimulation (FIG. 2a, lower trace). The time course of EPSC amplitude evoked by 40 stimuli at 14 Hz in both wild type and mutant mice was summarized (Figure 2b). The relative EPSC amplitude at the end of the sequential stimulation was 148 ± 13% (π = 13) and 207 ± 23% (η = 16) of the first EPSC in wild-type and mutant mice, respectively. The difference between the two groups was significant at all time points (p <0.05, t-test) (Figure 2b, star ). Since the phenotype of this Rab GDIa-deficient mouse is clearly opposite to the phenotype of Rab3A-deficient mouse (Ref. 18), the proposed function of Rab3A, namely, exocytosis during repeated stimulation! Suggests that synaptic vesicle recruitment is enhanced in Rab GDIα-deficient mice.

2.3 Enhancement of PPF in Rab GDIa-deficient mice

In the case of Rab3A-deficient mice, the continuous EPSC amplitude was virtually identical between wild-type and mutant mice during the first 12 stimuli (Ref. 18), and Rab3A was vesicular only after the 13th stimulus. It has been shown to be involved in replenishment. In contrast, the difference was significant in this study, even from the second stimulus (Figure 2b). This increased the likelihood that synaptic facilitation was altered in Rab GDI α? -Deficient mice. To test this possibility, another form of presynaptic plasticity, PPF, was tested. As expected from the results in Figure 2b, the amplitude of PPF was greater in Rab GDIa-deficient mice than in wild-type mice (Figure 3a). The ratio of EPSC amplitude at the second time to the first time at the 50 ms interval was 186 ± 6% (n = 17) and 216 ± 7% (n = 15) in wild-type and mutant mice, respectively (Fig. 3b). Differences between the two groups were significant at intervals of 50 ms and 70 ms (* P <0.05, t-test). On the other hand, PPF at longer intervals (100 ms, 200 ms and 300 ms) was not different between these two groups. The above result that PPF enhancement in mutant mice was not related to changes over time indicates that the increase in neurotransmitter release was not solely due to the formation of Ca2 ⁺ ions.The absence of Rab GDIa It is also due to changes in the intrinsic properties of the synaptic vesicles below. This result, together with reduced synaptic suppression during repetitive stimulation, suggests that the mechanism underlying this form of short-term plasticity may be dependent on Rab3A-mediated synaptic vesicle trafficking. I have.

2.4 No significant difference in LTP between wild-type and Rab GDIa-deficient mice

Finally, it was tested whether LTP in the CA1 region, an NMDA receptor-dependent form of synaptic plasticity, was altered in Rab GDIα-deficient mice. LTP was essentially identical in the two groups. That is, the wild type was 148 ± 11% (η = 9) of the baseline 60 minutes after induction, and the knockout mouse was 155 ± 19% (n = 7; P> 0.7, t-test) (Fig. Four). CA1 area Since LTP was not affected in Rab3A-deficient mice according to reference 18, this form of long-term plasticity appears to be essentially independent of Rab3A function. Industrial applicability

As described in detail above, the invention of this application provides an animal individual that is specifically expressed in nerve tissue and has a genetic deficiency of Rab GDIα, which is a causative gene of a neurological XLMR. It is useful for elucidating molecular mechanisms related to memory and learning, or for diagnosing neurological diseases such as epilepsy and developing therapeutic methods and therapeutics. Further, the Rab GDI α gene-deficient animal of the present invention is also useful as a model animal for a neurological disease XLMR. References

1. Von Gersdorff, H. & Matthews, G. Electrot Jhysiology of synaptic vesicle cycling. Annu. Rev. Physiol. 61, 725-752 (1999).

2. Zucker, R.S.Short-term synaptic plasticity. Annu. Rev. Neurosci. 12, 13-31 (1989).

3. Bliss, T.V.P. & collingridge, G.L.A synaptic model of memory: long-term potentiation in the hippocampus.Nature 361, 31-39 (1993).

4.Sasaki, T. et al. Purification and characterization from bovine brain cytosol of a protein inhibiting the dissociation of GDP from and the subsequent binding of GTP to smg p25A, a ras p21-like GTP- omding protein.J. Biol. Chem. 265, 2333-2337 (1990).

5. Matsui, Y. et al. Molecular cloning and characterization of a novelty e of regulatory protein (GDI) for smg p25A, a ras p21-like GTP-binding protein. Mol. Cell. Biol. 10, 41 16-4122 (1990).

6. Araki, S. et al. Regulation of reversible binding of smg 25A, a ras p21- like GTP-omding protein, to synaptic membranes and vesicles by its specific regulatory protein, GDP dissociation inhibitor. J. Biol. Chem. 265, 13007- 13015 (1990). 7. Pfeffer, SR, Dirac-Svejstrup, AB & Soldati, T. Rab GDP dissociation inhibitor: putting Rab GTPase in the right place. J. Biol. Chem. 270, 17057-17059 (1995).

8. Takai, Y ', Sasaki, Τ, Shirataki, H. & Nakanishi, H. Rab3A small GTP- omding protein in Ca ² "-dependent exocytosis. Genes Cells 1, 615-632

(1996).

9. Wu, S-K., Zeng, Κ, Wilson, I.A. & Balch, W. EStructural insights into the function of the Rab GDI superfamily. Trends Biochem. Sci, 21, 472-476 (1996).

10. Novick, P. & Zerial, M. The diversity of Rab proteins in vesicle transport.

Curr. Opin. Cell Biol. 9, 496-504 (1997).

1 1. Bachner, D. et al. Expression patterns of two human genes coding for different rab GDP- dissociation inhibitors (GDIs), extremely conserved proteins involved in cellular transport.Hum. Mol. Genet. 4, 701-708 (1995) .

12. D'Adamo, P. et al. Mutations in GDI l are responsible for X-linked nonspecific mental retardation. Nat. Genet. 19, 134-139 (1998).

13. Verkerk, A. J. et al. Identification of a gene (FMR-1) containing a CGG repeat coincident with a breakpoint cluster region exhibiting length variation in fragile X syndrome.Cell 65, 905-914 (1991).

14. Allen, K.M. et al. PAK3 mutation in nonsyndromic X-linked mental retardation. Nat. Genet. 20, 25-30 (1998).

15. Billuart, P. et al. Oligophrenin-1 encodes a rhoGAP protein involved in X- linked mental retardation.Nature 392, 923-926 (1998).

16. Sedlacek, Z. et al. Evolutionary conservation and genomic organization of XAP-4, an Xq28 located gene coding for a human rab GDP- dissociation inhibitor (GDI). Mamm. Genome 5, 633-639 (1994).

17. Armfield, K. et al. X-linked mental retardation syndrome with short stature, small hands and feet, seizures, cleft palate, and glaucoma is linked to Xq28. Am. J. Med. Genet. 85, 236-242 (1999).

Geppert. M. et al. The role of Rab3A in neurotransmitter release.Nature 369, 493-497 (1994).

Luan, P. Balch, W. Ε. 'Emr, S. D. & Burd, C. G. Molecular dissection of guanine nucleotide dissociation inhibitor function in vivo.J. Biol. Chern. 274, 14806-14817 (1999).

Traub, R.D., Miles, R. & Wong, R.K.S.Model of the origin of rhythmic population oscillations in the hippocampal slice.Science 243, 1319-1325 (1989).

Koera, K. et al. K-ras is essential for the development of the mouse embryo.Oncogene 15, 1 151-1159 (1997).

Claims

The scope of the claims

1. Rab GDI α gene, which is a non-human animal that has generated totipotent cells in which the genomic gene encoding the low molecular weight GTP-binding protein Rab GDI α has been replaced by a mutant gene with a loss of function, and its progeny Deficient animal.

2. The Rab GDIa gene-deficient animal according to claim 1, wherein the non-human animal is a mouse.

3. A tissue or cell derived from the animal of claim 1 or 2.

4. ES cells in which the genomic gene encoding the low-molecular-weight GTP-binding protein Rab GDI or has been replaced by a mutant gene lacking its function.

5. The ES cell according to claim 4, which is derived from a mouse.