JP4374438B2 - lab8a gene-deficient mouse - Google Patents

Description

  The present invention relates to a rab8a gene-deficient mouse that is useful as a model of human nutrient absorption disorder.

Epithelial cells covering the surfaces of digestive organs, skin, urinary organs, respiratory organs, etc. have directionality (polarity) on the apical side and basolateral side. Neurons also have axons and dendrites. The normal formation of polarity is essential for the normal development and secretion of tissues and organs and functions such as transmission of stimuli. In order to form and maintain polarity, it is necessary to synthesize and secrete secreted proteins and membrane proteins into transport vesicles by trans-Golgi network and then transport them to the apical and basolateral sides. This directional transport is called polar transport. It is known that low molecular weight GTP-binding protein, SNARE protein, etc. are important for polar transport.
As one of low molecular weight GTP-binding proteins, a group of Rab molecules is known. These molecules have an active GTP binding type and an inactive GDP binding type. The GDP binding type exists mainly in the cytoplasm, and the GTP binding type exists in the membrane (cell membrane or intracellular membrane). Rab is known to perform its function by moving between the cytoplasm and the membrane. Currently, more than 60 types of Rab molecules are known, but each molecule has a unique distribution in the cell and is thought to function in each location.
Rab8a has been considered to be necessary for transporting vesicles formed by the trans-Golgi network, which is the exit of proteins modified with the Golgi apparatus, to the basolateral side (Non-patent Document 1). However, no mouse lacking the rab8a gene has been reported, and the function of the Rab8a protein has not been fully elucidated.

Microvilli atrophy is a nutrient-absorbing disorder in which the microvilli on the cell surface cannot sufficiently absorb nutrients, cause diarrhea immediately after birth, and exhibit dehydration and malnutrition (Non-patent Document 2). ). There are many refractory absorption disorders in children, such as microvillous atrophy, and small intestine transplantation has mainly been used as the fundamental treatment. However, transplantation involves a lot of cost and ethical issues, so it is difficult to say that transplantation is actively performed in Japan. Also, because of its high cost, it is not a treatment that can be applied to anyone. However, it is impossible to use humans for the development of other therapies, and a model animal that reproduces the pathology well is required.
J Cell Biol. 1993 Oct; 123 (1): 35-45. Clin Gastroenterol 1986 Jan; 15 (1): 105-20

  An object of the present invention is to provide a model animal that can be used for screening of therapeutic agents for nutrient absorption disorder diseases and development of therapeutic methods.

  The present inventor has intensively studied to solve the above problems. As a result, it was found that mice lacking the rab8a gene on the chromosome cause microvillous atrophy in the small intestine, jejunum, etc., and exhibit nutrient absorption disorders, thereby completing the present invention.

That is, the present invention is as follows.
(1) A rab8a gene-deficient mouse in which the function of the rab8a protein is deleted by replacing the rab8a gene on the chromosome with an inactive rab8a gene.
(2) The rab8a gene-deficient mouse according to (1), wherein the inactive rab8a gene is a rab8a gene lacking exon 2.
(3) The rab8a gene-deficient mouse according to (1) or (2), which is a model mouse for a nutrient absorption disorder disease.
(4) A compound of a nutrient absorption disorder characterized by administering a compound to any one of (1) to (3) or a cell obtained from the mouse, and evaluating the effect of the compound on improving nutrient absorption disorder A screening method for therapeutic drugs.
(5) A method for analyzing a nutrient absorption disorder characterized by using the mouse of any one of (1) to (3) or a cell, tissue or organ obtained from the mouse.

In the rab8a gene-deficient mice of the present invention, microvilli atrophy and reduced nutrient absorption occur, so using this mouse causes diseases that cause reduced human nutrient absorption, such as microvillus atrophy, short bowel syndrome, The mechanism of diseases such as Hirschsprung's disease can be elucidated. It is also possible to screen for drugs for the treatment of these diseases by administering drugs or the like to these mice. Furthermore, gene therapy can be performed by introducing a rab8a gene from the intestinal tract using a viral vector or the like.

The present invention is described in detail below.
The rab8a-deficient mouse of the present invention (rab8a knockout mouse) is a mouse that lacks the function of the rab8a protein by replacing the rab8a gene on the chromosome with an inactive rab8a gene. "Inactive rab8a gene" refers to deletion of part of the rab8a gene, insertion of other nucleotide sequences into the coding region of the rab8a gene, point mutations within the rab8a gene, mutations within the expression regulatory region of the rab8a gene, etc. Refers to a gene that cannot express normal rab8a protein. Examples of the defective rab8a gene include, but are not limited to, a gene lacking exon 2. “Deficient in rab8a protein function” means that the function of rab8a protein, such as vesicle transport, has been lost, and preferably means that rab8a protein function has been completely lost. This includes cases where only one of the alleles is replaced with an inactive form, such as a mouse, and the function of the rab8a protein is partially lost.

  Examples of the rab8a gene include a gene encoding the amino acid sequence of SEQ ID NO: 2, more specifically, the mouse rab8a gene having the base sequence represented by SEQ ID NO: 1. This base sequence is registered in GenBank with the accession number BC019990. Moreover, since the rab8a gene varies depending on the species, it may be the homologous gene of SEQ ID NO: 1. The homologous gene is homologous enough to cause homologous recombination with the rab8a gene on the mouse chromosome, for example, 80% or more, preferably 90% or more, more preferably 95% or more homology with the nucleotide sequence of SEQ ID NO: 1. The thing which has property is mentioned. In addition, the homologous gene may be a gene that hybridizes with the gene having the base sequence of SEQ ID NO: 1 under stringent conditions. Here, as stringent conditions, for example, hybridization is performed, and 65 ° C., 1 × SSC, 0.1% SDS, preferably 65 ° C., 0.1 × SSC, 0.1% SDS. The conditions to wash | clean are mentioned.

The rab8a gene-deficient mouse of the present invention can be prepared by a known gene recombination method (gene targeting method). For example, it can be produced as follows.
First, a partial fragment of the rab8a gene is prepared, and a targeting vector for replacing the rab8a gene with a defective type is prepared.
For selection of recombinants, it is preferable to incorporate a drug resistance gene into the targeting vector. A neomycin resistance gene, a hygromycin B phosphotransferase gene, or the like can be used as a marker gene for drug selection.
In addition, in order to delete a partial sequence of the rab8a gene, the Cre-loxP system (R. Kuhn et al. Science, 269, 1427-1429, 1995) and the FLP / FRT system (Rodriguez et al. Nat Genet 25: 139 -40.) May be used. In that case, a targeting vector is constructed so that the partial sequence to be deleted is sandwiched between loxP sequences or FRT sequences.

Hereinafter, a general method for obtaining a rab8a gene-deficient mouse using the above-described targeting vector will be described. However, the mouse of the present invention is not limited to that obtained by the following method.
Homologous recombination is performed using the targeting vector prepared by the above method. For homologous recombination, a method using embryonic stem cells (ES cells) can be used. Several mouse-derived ES cell lines have been established, and TT2 cell line, AB-1 cell line, J1 cell line, R1 cell line, E14TG2a cell line and the like can be used. The targeting vector can be introduced into mouse ES cells according to a known method. For example, an electroporation method, a liposome method, a calcium phosphate method, a DEAE-dextran method, and the like can be used.
Next, a cell clone in which the wild-type rab8a gene on the chromosome is replaced with the defective rab8a gene in the targeting vector is selected. Selection can be performed based on drug resistance and the like, and it is preferable to confirm homologous recombination by Southern blotting or PCR.

  ES cells having the mutated gene thus obtained are introduced into embryos of wild-type mice. Then, a chimeric animal can be produced by transplanting this ES cell embryo into the uterus of a foster mother of a pseudopregnant state and giving birth. As a method for introducing ES cells into an embryo such as a blastocyst, a microinjection method and an aggregation method are known, but the microinjection method is more preferable. A pseudopregnant female mouse for use as a foster parent can be obtained by mating a female mouse having a normal cycle with a male mouse castrated by vagina ligation or the like.

The chimeric mouse is then mated with a pure-line mouse, and the ES cells are transferred to the germ line of the chimeric mouse. A heterozygote lacking the rab8a gene can be obtained by selecting an animal in which the recombinant ES cell transplanted into the embryo has entered the germ line and breeding the animal.
By mating the obtained rab8a gene-deficient heterozygous mice, a rab8a gene-deficient homozygous mouse can be obtained.

  In preparing the knockout mouse of the present invention, the above-mentioned Cre / loxP system and FLP / FRT system, which are widely used in the production of a conditional knockout mouse, can also be used. When using the Cre / loxP system, by crossing with an animal expressing Cre recombinase, which is a recombination enzyme derived from P1 phage of Escherichia coli, or by infecting a viral vector having a gene having the Cre gene, Cre recombinase can create a rab8a gene-deficient mouse to recognize and remove the sequence flanked by loxP sequences. In addition, a knockout having a characteristic that a gene is deficient in a tissue-specific manner can be produced by mating with a mouse expressing a Cre enzyme in a tissue-specific manner. Similarly, when the FLP / FRT system is used, a rab8a gene-deficient mouse can be produced by mating with an animal expressing FLP recombinase.

The rab8a gene-deficient mouse produced in this way causes microvilli atrophy, impaired protein transport to the apex, etc., and exhibits a nutrient absorption disorder such as malnutrition.
Therefore, by using this mouse, or a mouse-derived cell, tissue, or organ, diseases that cause a decrease in human nutrient absorption, such as microvilli atrophy, short bowel syndrome, Hirschsprung's disease, etc. Elucidation can be done. In addition to these diseases, it is possible to elucidate the onset mechanism of various diseases related to rab8a.

It is also possible to screen for therapeutic agents for various diseases associated with rab8a. For example, by administering a compound to a rab8a gene-deficient mouse or a cell obtained from the mouse and evaluating the effect of improving the nutrient absorption disorder of the compound, a therapeutic drug for a nutrient absorption disorder can be screened. The effect of improving nutrient absorption disorder may be evaluated directly by absorption of glucose or the like, or may be evaluated by the function of molecules involved in nutrient absorption.
Furthermore, since it is considered that the rab8a gene can be introduced from the intestinal tract using a viral vector or the like to recover the symptoms, it can also be used as a model for gene therapy via the intestinal tract.

<Production of knockout mouse>
A 15 kb fragment containing the rab8a gene was isolated from a genomic library derived from 129 / Sv mouse and incorporated into pBlueScript (+) (Stratagene). SA-IRES-βgeo pA (Homma et al), a cassette for gene disruption consisting of SA (splice acceptor site of bcl2 gene), IRES (internal ribosomal entry site), and βgeo (fusion gene of β-galactosidase gene and neomycin resistance gene) Cell. 2003 Jul 25; 114 (2): 229-39.), The DNA construct obtained by linking the FRT site before and after that and the loxP site on the 5 ′ side is the second of the rab8a gene. It was inserted into the BamHI site in the intron so that the direction of transcription was the same. Another targeting site was obtained by inserting another loxP site into the XhoI site present in the first intron (FIG. 1).
Next, the obtained targeting vector was introduced into ES cells by electroporation. ES cells of the J1 line were used and cultured and electroporated according to the method described in Joyner et al. (Gene Targeting: Practical Approach. IRL Press, New York, p234, 1993). ES cells were cultured in a neomycin-containing medium, transformants were selected and cell lined, and homologous recombination to the targeting vector was confirmed by Southern blotting.
ES cells into which the targeting vector was integrated on the chromosome were injected into blastocysts of C57BL / 6 mice. A blastocyst injected with ES cells was transplanted into a foster parent mouse that had been pseudopregnant, and a baby was born to obtain a chimeric mouse.
This chimeric mouse was crossed with a wild type mouse to obtain a heterozygote. Next, the heterozygote was crossed with a transgenic mouse (B6; SJL-Tg (ACTFLPe) 9205Dym / J; Jackson Laboratory) expressing the FLP recombination enzyme to remove SA-IRES-βgeo pA. Furthermore, the obtained mouse was crossed with a transgenic mouse (CAG-Cre mouse) expressing Cre throughout the body to obtain a rab8a (+/−) mouse lacking the second exon of the rab8a gene. Furthermore, this rab8a (+/−) mouse was crossed to obtain a rab8a gene completely deficient mouse (rab8a (− / −) mouse). As a result of examining the rab8a gene-deficient mice, it was found that they had a nutrient absorption disorder, and most of them died of malnutrition after 3 weeks of age.
Furthermore, the following analysis was performed using these mice.

<Immunohistochemical staining>
2.5 week old rab8a (+/-) and rab8a (-/-) mice were anesthetized with ether and Nembutal and perfused with 3% paraformaldehyde / 0.1 M phosphate buffer (pH 7.4). Various organs such as brain, liver, small intestine, kidney and pancreas were excised and stored in fixative overnight at room temperature. The procedures of cryoprotection, freezing, and section preparation were performed according to the method described in Harada et al. (Cell Struct. Funct. 15 (6), 329-342, 1990). Among these, sections of the small intestine were examined for expression distribution of SGLT (Na-glucose transporter) and DPP4 (dipeptidyl peptidase 4) by fluorescent immunohistochemical staining. The tissue section was first reacted with a primary antibody (goat anti-SGLT antibody (Santa Cruz Biotechnology, INC.) Or goat anti-DPP4 antibody (R & D Systems)), then secondary antibody (Alexa 488-conjugated donkey anti-goat IgG; (Molecular Probes). Observation was performed using a scanning confocal microscope (model MRC1024; Zeiss).
As a result, SGLT and DPP4 were found on the apical side as usual in rab8a (+/-) mice, whereas SGLT and DPP4 were reduced from the apical side in rab8a (-/-) mice and incorporated into the cells. (Figure 2). This was unexpected from the theory that rab8a has been proposed so far to transport vesicles to the basolateral side.

<Observation by electron microscope>
Perfuse 3 week old normal mice (rab8a (+ / +)) or knockout mice (rab8a (-/-)) with 2% paraformaldehyde, 2.5% glutaraldehyde / 0.1M cacodylate buffer (pH 7.4), The small intestine, liver and kidney were removed and fixed for another 2 hours. Among these, the jejunum sections were observed with an electron microscope. Jejunal sections were prepared according to the method described in Harada et al. (Cell Struct. Funct. 15 (6), 329-342, 1990) and observed under a voltage of 80 kV using a JEOL1010 electron microscope. As a result, it was found that rab8a (-/-) showed atrophy of microvilli and that some of the microvilli were taken up inside the cells.
This phenomenon was very similar to the findings of microvillous atrophy, which is one of the human diseases that causes diarrhea immediately after birth, causes dehydration and malnutrition, and is fatal if not treated.

Schematic diagram of rab8a gene disruption. The figure which shows the expression of SGLT and DPP4 in the small intestine of a rab8a gene hetero knockout mouse (+/-) or a homo knockout mouse (-/-) (photograph; magnification 100 times). The figure which shows the observation result by the electron microscope of the jejunum of a normal mouse or a rab8a gene homo knockout mouse (-/-) (photograph; magnification 5000 times, lower right further enlarged figure).

Claims (4)

By replacing the rab8a gene on the chromosome with the inactive rab8a gene , the compound is administered to a rab8a gene-deficient mouse lacking the function of the rab8a protein or cells obtained from the mouse, and the effect of improving the nutrient absorption disorder of the compound is improved. A screening method for a therapeutic agent for a nutrient absorption disorder characterized by evaluating. The method according to claim 1, wherein the inactive rab8a gene is a rab8a gene lacking exon 2 . Nutrient absorption characterized by using a rab8a gene-deficient mouse deficient in the function of the rab8a protein or a cell, tissue or organ obtained from the mouse by replacing the rab8a gene on the chromosome with an inactive rab8a gene Method for analyzing disability disease. The method according to claim 3, wherein the inactive rab8a gene is a rab8a gene lacking exon 2 .

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