KR101289474B1 - Transgenic Mouse Useful for Researching the Function of Ephrin-A5 Gene and the Method For Preparing the Same - Google Patents

Abstract

The present invention discloses transgenic mice useful for ephrin-A5 gene function studies and methods of making the same. Specifically, the present invention includes a reporter gene whose expression is regulated by a promoter and enhancer of ephrin-A5 and an ephrin-A5-Fc gene located below the reporter gene, wherein the reporter gene is removed by homologous recombination. By including a recombinant sequence at both ends of the reporter gene so that it can be identified, the region expressed by the expression of the reporter gene Ephrine-A5 gene can be identified, when the reporter gene is removed by homologous recombination Ephrin-A5 Expression of the -Fc gene results in loss or degradation of Ephrine-A5 by binding Ephrine-A5-Fc to the Eph receptor to which Ephrine-A5 binds, resulting in loss or degradation of A5. Disclosed are a transgenic mouse and a method of making the same, which make it possible to identify a phenotype according to the present invention.

Description

Transgenic Mouse Useful for Researching the Function of Ephrin-A5 Gene and the Method For Preparing the Same}

The present invention relates to transgenic mice useful for ephrin-A5 gene function studies and methods of making the same.

Genetic targeting techniques have suggested that the interaction of the ephrin-A5 ligand with the EphA7 receptor acts as a zipper that promotes neural folds in neural tube formation in the mouse hepatic and midbrain (Holmberg, et al., Nature 408: 203-6.).

To date, about 15 Eph receptors and about 9 ephrin ligands have been found in vertebrates, which are classified into A type and B type, respectively. Type A Eph receptors specifically bind to type A ephrin ligands, and type B Eph receptors are known to bind specifically to type B ephrin ligands. In addition, receptors and ligands in the same subgroup are very similar to each other in specific binding, and it is known that several Eph / ephrine genes are expressed together in the same tissue. Therefore, mice hitting either the Eph or ephrin genes rarely have a fatal effect on embryo development and survival, whereas mice hitting two or more genes simultaneously die during embryonic development. It has been reported. These results suggest that different types of Eph receptor / ephrin ligands are expressed in function duplication during mouse development.

This duplication of Eph receptor / ephrine ligands is a major obstacle to the study of EphA7 / Ephrine-A5 gene expression regulators that regulate neural tube junctions and the Eph7 / Ephrine-A5 signaling system that promotes neural junctions. come.

To date, research on EphA7 / Ephrine-A5 has shown that EphA7 / Ephrine-A5 is specifically expressed in cells in the dorsal midline of mouse liver and midbrain (Holmberg, et al., Nature 408). Mice that do not express the EphA7 / Ephrin-A5 gene by gene targeting techniques show cerebral anemic disease at 5% and 30%, respectively (Holmberg, et al., Nature) . 408: 203-6.).

For genes that play a very important role in development, tissue or cell-specific gene knock-out techniques are more effective in analyzing gene function than conventional gene targeting techniques (Falk, et al., Cell Stem Cell 2: 472-83.).

It is an object of the present invention to provide a transgenic mouse useful for studying the function of ephrin-A5 by making it possible to lose or decrease the function of ephrin-A5 in tissue or cell specificity.

Another object of the present invention to provide a method for producing the transgenic mouse.

Other and specific objects of the present invention will be presented below.

In one aspect, the invention relates to a transgenic mouse useful for studying the function of ephrin-A5 by making it possible to lose or reduce the function of ephrin-A5 in tissue or cell specificity.

The present inventors first isolated the enhancer factor of the ephrin-A5 gene for the production of transgenic mice useful for studying the function of ephrin-A5.

Specifically, in order to separate the enhancer factor of the ephrin-A5 gene, the present inventors first identified four bacterial artificial chromosome (BAC) clones containing the first exon of the ephrin-A5 gene, that is, -207.5 kb to +11.6 kb ( RPCI-23-4O15, a BAC containing the base sequence of the first nucleotide of the first exon, based on + lb, RPCI-23-375B7, which contains the base sequence of -154.4kb to + 57.7kb RPCI-23-211L12 containing a nucleotide sequence of 71.5kb to + 115.3kb, and RPCI-23-23O22 containing a nucleotide sequence of -6.7kb to + 214.8kb were purchased from Children's Hopspital Oakland Research Institute, and each of the above BACs The lacZ gene was inserted by homologous recombination using a targeting vector in the first exon of the mouse, and each BAC containing the lacZ gene was injected into a mouse fertilized egg and transplanted into the surrogate mother, followed by development 8.5, 9.5 and 10.5. X by separating mouse embryos from day one When the -gal staining, except for the RPCI-23-4O15-inserted mice, all of the other BAC-inserted mice were confirmed that the center and midbrain centerline staining.

From these experimental results, the inventors confirmed that the enhancer of the ephrin-A5 gene is present between + 11.6kb and + 57.7kb, and removed the + 0.2kb to + 30.5kb region (30.3kb size) in order to further narrow the range. The target vector was prepared, and the region was removed from RPCI-23-375B7 by homologous recombination, and RPCI-23-375B7del30.3, a BAC from which the site was removed, was inserted into the mouse embryo and generated through surrogate mothers. After mouse X-gal staining, it was confirmed that hepatic and midbrain centerlines were not stained. These experimental results show that the enhancer of the Ephrin-A5 gene is present between + 11.6kb and + 30.5kb. The present inventors further divided the interval into three fragments of 5-7kb size (the first fragment is +11.6). fragments from kb to +18.8 kb, second fragment from +18.1 kb to +25.1 kb, third fragment from +25.0 kb to +30.5 kb), and each fragment divided into ephrin- When the transgenic mouse was constructed using the recombinant vector coupled with the promoter of A5 gene and lacZ gene, it was confirmed that X-gal staining occurred in the embryonic liver and midbrain centerlines only when the third fragment was inserted. .

In summation of the experimental results described above, the enhancer factor of the Ephrin-A5 gene specific for the hepatic and midbrain centerlines is the third fragment from + 11.6kb to + 30.5kb, that is, from + 25.0kb to + 30.5kb. It can be seen that. The base sequence of the third fragment is shown in SEQ ID NO: 1.

Thus, after separating the enhancer factor of ephrin-A5, a target vector capable of homologous recombination to a BAC containing the enhancer factor of ephrin-A5 was prepared, and the recombinant vector was the first exon sequence of the ephrin-A5 gene. Homology A cancer (SEQ ID NO: 2) and B cancer (SEQ ID NO: 3) constructed based on both terminal sequences of the first exon of ephrin-A5 to be recombined into In between, an enhanced green fluorescent protein gene (eGFP) gene and a polyadenylation signal sequence, a reporter gene with a loxP sequence at both ends, a recombination site, ephrin-A5-Fc gene and polyadene under the eGFP gene It was constructed to include a kanamycin resistance gene having a nilation signal sequence and a FRT sequence, a recombinant sequence at both ends, under the ephrin-A5-Fc gene.

Homologous A cancer in the above is a sequence from -293b to + 146b based on the start position (+ 1b) of the first exon, this portion is Ephrin-A5 when referring to the Reference Examples and Examples below It acts as a promoter of.

In the recombinant sequences of the loxP sequence in the Cre recombinase of bacteriophage P1 (Hoess and Abremski (1990) In Nucleic Acids and Molecular Biology, vol 4. Eds .: Eckstein and Lilley, Berlin-Heidelberg:. Springer-Verlag; pp. 90-109) is a recombinant sequence specifically recognized, and the FRT recombinant sequence is Saccharomyces cerevisiae ) Flp recombination enzyme (Broach, et al., Cell 29: 227-234 (1982)) is specifically recognized as a recombinant sequence.

The prepared target vector was a BAC containing an enhancer factor of ephrin-A5 and comprising a first exon sequence of ephrin-A5 (ie, RPCI-23-375B7, RPCI-23-211L12 and RPCI-23-23O22) Recombinant RPCI-23-23O22 using homology A and B cancer, transformed FLP plasmid capable of expressing Flipase to induce FRT recombination sequence dependent homologous recombination to remove the kanamycin resistance gene, finally RPCI-23 recombined between A and B cancers, the eGFP gene, a reporter gene with a loxP sequence at both ends, a polyadenylation signal sequence, and the ephrin-A5-Fc gene and a polyadenylation signal sequence beneath the eGFP gene. -23O22 was produced. The recombinant RPCI-23-23O22 originally contains an enhancer factor of RPCI-23-23O22.

Next, when the DNA of the recombinant RPCI-23-23O22 was isolated and microinjected into the fertilized egg of the mouse and the fertilized egg was generated, the embryo of the transgenic mouse was observed by fluorescence microscopy, and the reporter gene eGFP was ephrin-A5 gene. It can be confirmed that the expression region in the center line of the brain, middle brain and middle brain.

Furthermore, Wnt1 gene (Danielian, et al., Curr , which is known to express tissue-specifically along the nerve junction region of the transgenic mouse) Biol . 8: 1323-6) and mice transfected with Cre genes regulated by the Wnt1 enhancer factor and observed from day 9.5 of embryonic development, the brain size became smaller and two cranial faces It was found that it was not properly developed. As eGFP gene was cleaved as a result of cleaving the reporter gene eGFP by Cre recombination enzyme, the next gene, ephrin-A5-Fc gene, was expressed and ephrin-A5. It is believed that -Fc binds to the Eph receptor and eventually blocks the binding of Ephrine-A5 to the Eph receptor.

The present invention is provided based on the results of these experiments. The transgenic mouse of the present invention is a promoter of ephrin-A5, a reporter gene located downstream of the promoter and having a recombinant sequence at both ends and the 3 'of the gene. A regulatory sequence, an ephrin-A5-Fc gene located below the reporter gene and a 3 'regulatory sequence of the gene, and an enhancer of ephrin-A5 located below the ephrin-A5-Fc gene The mouse transformed by the recombinant expression vector.

As used herein, "transformation" means that exogenous DNA is introduced into a host organism (mouse in the present invention) such as a cell, tissue, or adult, stably inherited, and exhibits a change in phenotype. Exogenous DNA introduced into such host organism can be inserted into the genome of the host organism.

Since the transgenic mouse of the present invention is configured to regulate the expression of the reporter gene (and the ephrin-A5-Fc gene) by the ephrin-A5 promoter and the ephrin-A5 enhancer factor, the transformed mouse is ephrin-. The reporter gene is expressed in the region where the A5 gene is expressed, and as a result, the region where the ephrin-A5 gene is expressed can be identified by separating the embryo during development and confirming the expression region of the reporter gene.

In addition, since the transgenic mouse of the present invention contains a recombinant sequence, when the recombinant mouse is crossed with a mouse transformed with a gene of a recombinant enzyme capable of recognizing the recombinant sequence and cleaving a reporter gene between the recombinant sequences, In generation mice (including embryos), as the reporter gene is cleaved, ephrin-A5-Fc gene is expressed so that ephrin-A5-Fc binds to Eph to which ephrin-A5 binds, or the function of ephrin-A5 is lost or It becomes possible to reduce, and as a result, the phenotype according to the loss or fall of the function of ephrin-A5 can be confirmed.

In the transgenic mouse of the present invention, the "promoter of ephrin-A5" coincides with the sequence of homology A cancer as described above, and from -293b based on the starting position of the first exon (+ 1b) Refers to sequences up to + 146b. This promoter sequence is disclosed in SEQ ID NO: 2. Homologous A cancer can be seen to function as a promoter of ephrin-A5 in the following Reference Examples and Examples.

As used herein, a "promoter" is usually located above a translational initiation codon of a gene and includes one or more binding sites for DNA-dependent RNA polymerase, transcriptional initiation sites, binding sites for proteins involved in transcriptional initiation, and the like. A nucleic acid sequence having a function of controlling transcription of a gene is meant.

In addition, in the present specification, the meaning of “upper” refers to a sequence located at 5 ′ of the reference nucleotide sequence, and in this regard, the term “lower” refers to a nucleotide sequence located at 3 ′ of the reference nucleotide sequence. For example, in general, a translation initiation codon of a gene may be located below it based on a transcription initiation site, and conversely, a transcription initiation codon may be located above it based on a translation initiation codon of a gene.

In the transgenic mouse of the present invention, the reporter gene refers to a gene whose expression can be analyzed, and any such gene can be used. Examples of such reporter genes include green fluorescent protein (GFP) gene, red fluorescent protein (RFP) gene, luciferase gene, GUS gene, lacZ gene, CAT (chloramphenicol acetyltransferase) gene and the like. Analysis of the expression of such reporter genes may use any method known in the art suitable for each reporter gene, and such methods include immunochemical methods, enzymatic methods, methods by fluorescence detection, and the like.

In the transgenic mouse of the present invention, the ephrin-A5-Fc gene is a gene specified by SEQ ID NO: 4, and the ephrin-A5-Fc protein, which is an expression product of the gene, is ephrin-A5 (GenBank Accession No. NP_997537.1) is a fusion of an extracellular region of human IgG1 Fc sequence (GenBank Gene ID: 3500, Product Accession NO. P01857). The fusion proteins have the ability to combine the Eph receptor which combines a F Lin -A5 can block signal transduction by F. Lin -A5 (Flanagan and Vanderhaeghen, Annu Rev Neurosci 21:... 309-45. ;.. Pasquale, Curr Opin Cell Biol . 9: 608-15.). The sequence of the fusion protein is set forth in SEQ ID NO: 19, wherein the 203th amino acid Asn is a sequence derived from ephrin-A5, and the 204th amino acid Pro is a sequence derived from Fc of human IgG1.

Such ephrin-A5-Fc genes can be synthesized or commercially available from R & D systems: Catalog Number: 374EA: Recombinant Human EphrinA5 Fc Chimera.

In the transgenic mouse of the present invention, the reporter gene or the ephrin-A5-Fc gene is regulated by the ephrin-A5 promoter and the ephrin-A5 enhancer factor. Regulatory sequences are operably linked.

The 3 ′ regulatory sequence refers to a sequence that is capable of affecting terminator sequences, polyadenylation signal sequences, and other mRNA processing or gene expression as expression control sequences located below genes operably linked thereto. Such 3 ′ regulatory sequences may be derived from a variety of hosts, such as SV40 polyadenylation signal, bovine growth hormone (BGH) polyadenylation signal, viral terminator sequences, and the like. Such 3 ′ regulatory sequences may be artificial sequences derived from these native sequences.

In the transgenic mouse of the present invention, the expression vector may be a plasmid or BAC used in the following examples of the present invention, or may be a virus-derived vector. In the following examples of the present invention, RPCI-23-23O22 of RPCI-23-375B7, RPCI-23-211L12 and RPCI-23-23O22 containing an enhancer factor of the ephrin-A5 gene was used, but RPCI-23- 375B7 or RPCI-23-211L12 may be used, or any BAC containing an enhancer factor of other ephrin-A5 genes may be used. Any BAC containing the enhancer factor of the ephrin-A5 gene can be searched and confirmed in databases such as GeneBank (NCBI) in the United States, DNA Data Bank of Japan (DDBJ) in Japan, and European Molecular Biology Laboratory (EMBL) in Europe. It may be retained and purchased from Children's Hopspital Oakland Research Institute.

The "expression vector" is designed so that the inserted nucleic acid sequence (ie, the sequence of the target gene to be expressed) can be expressed after transformation into a host organism (including a fertilized egg, embryo, adult) in the host organism (in the present invention). Say vector. Such expression vectors usually include a promoter, an enhancer factor, and the like, along with the cloned gene. The transformed expression vector in the transgenic mouse of the present invention includes a promoter of the ephrin-A5 gene and an ephrin-A5 enhancer factor. .

The transgenic mouse of the present invention, as described above, since the expression of the reporter gene is regulated by the enhancer of the ephrin-A5 promoter and the ephrin-A5, it identifies itself the region in which the ephrin-A5 gene is expressed. In addition to the usefulness, the expression of ephrin-A5-Fc gene is removed when the reporter gene is removed using a mouse transformed with a recombinant enzyme gene that specifically recognizes the recombinant sequence and removes the gene between the recombinant sequences. Useful in the study of phenotype following loss or degradation of lean-A5 function.

In the following examples of the present invention, since the loxP sequence was used as the recombinant sequence, the recombinant enzyme Cre was used (that is, crossed with a mouse transformed with the Cre gene), but in addition to Cre, other sequence specific recombinant enzymes may be used. (Ie, may be crossed with mice transformed with other sequence specific recombinant enzyme genes). As such sequence-specific recombinase, yeast-derived recombinase FLP or Kw integrase (Buchholz et al, Nature Biotech., 16, 657-662 (1998); Ringrose et al, Eur. J. Biochem., 248, 903-912) , Integrase of bacteriophage lambda (Landy, A. Current Opinions in Genetics and Devel. 3: 699-707 (1993)), beta recombinase (β-recombinase), integrase of HK022 phage, phiC31 integrase (phiC31) integrase), lambda delta resolvase (Lorbach et al, J. Mol. Biol, 296, 1175-81 (2000); Kolot et al, Moi. Biol Rep. 26,207-213 (1999); Schwikardi et al FEBS Lett., 471, 147-150 (2000); Diaz et al, J. Biol Chem., 274, 6634-6640 (1999)).

Meanwhile, in the following examples, the transgenic mouse of the present invention was crossed with a mouse transformed with the Cre gene, which is a recombinant enzyme regulated by a promoter of the Wnt1 gene, which is known to be expressed along a neural junction region, but the recombinant enzyme Cre Is controlled by a promoter specific for other tissues or cells, the transgenic mouse of the present invention is crossed with a mouse transfected with a Cre gene regulated by such a promoter, and the expression of ephrin-A5 in that tissue or cell As the function is lost or decreased, the phenotype of the loss or degradation of ephrin-A5 in the tissue or cell can be examined.

As used herein, "tissue-specific promoter" means that the promoter selectively expresses a target gene operably linked to the promoter only in a specific tissue (such as the liver) and does not or does not express the target gene in other tissues (such as the brain). Refers to a promoter that expresses at a level.

Such tissue-specific promoters, in addition to the promoter of the WNT1 gene used in the examples of the present invention, neural tissue specific promoters such as the promoter of the nestin gene, the promoter of the Pax-1 gene, the promoter of the Engrailed-1 gene, Muscle specificity such as the promoter of albumin gene, the promoter of alpha-1 antirypsin gene, the promoter of myogenin gene, the promoter of actin gene, the promoter of MyoD gene, the promoter of myosin gene, etc. Testicular specific promoters such as oocyte-specific promoters such as red promoters, promoters of ZP1 gene, promoters of ZP2 gene, promoters of ZP3 gene, promoters of protamine gene, promoters of pertiline gene, promoters of globulin gene, GATA-1 Promoter of gene, porphobilinogen deamase blood-specific promoters such as inase) promoters, skin- or hair-specific promoters such as promoters of keratin genes and elastin genes.

In addition, the term "cell specific promoter" means that the promoter selectively expresses a target gene operably linked to the promoter only in specific types of cells, and does not express or lower the target gene in other types of cells of the same tissue. Refer to promoters that express at levels (Higashibata et al., J. Bone Miner. Res. Jan 19 (1): 78-88 (2004); Hoggatt et al., Circ. Res., Dec. 91 (12): 1151-59 (2002); Sohal et al., Circ.Res. Jul 89 (1): 20-25 (2001); Zhang et al., Genome Res. Jan 14 (1): 79-89 (2004).

Examples of such cell specific promoters include the vascular endothelial growth factor (VEGF) receptor (flk1) promoter (Kappel et al. Blood 93: 4282-4292 (1999)) expressed in endothelial cells, the insulin promoter expressed in the beta cells of the pancreas. (Ray et al., J. Surg. Res. 84: 199-203 (1999)), a promoter of the gonadotropin-releasing hormone receptor gene expressed in cells of the hypothalamus (Albarracin et al., Endocrinology 140: 2415 -2421 (1999)), matrix metalloproteinase 9 promoter expressed in osteoclasts and keratinocytes (Munant et al., J. Biol. Chem. 274: 5588-5596 (1999)), parathyroid glands expressed in bone cells Hormone receptors (Amizuma et al., J. Clin. Invest. 103: 373-381 (1999)), dopamine beta-hydroxylase promoters expressed in noradrenergic neurons (Yang et al., J. Neurochem. 71 : 1813-1826 (1998)).

The tissue specific promoter or cell specific promoter may be a constitutive promoter which induces expression constantly or an inducible promoter which induces expression in response to a specific external stimulus.

In another aspect, the present invention relates to a method for producing a transgenic mouse of the present invention as described above.

The method for producing a transgenic mouse of the present invention comprises (I) a promoter of ephrin-A5, a reporter gene whose recombinant sequence located downstream of the promoter, a 3 'regulatory sequence of the gene, and a reporter gene Constructing a recombinant expression vector comprising an ephrin-A5-Fc gene located downstream and a 3 'regulatory sequence of the gene, and an enhancer of ephrin-A5 located downstream of the ephrin-A5-Fc gene, and (II) introducing the recombinant expression vector into a mouse which is a host organism.

In the method for producing a transgenic mouse of the present invention, the step of preparing the recombinant expression vector may be performed by inserting each of the above factors into a plasmid vector or viral vector using a method known in the art, but preferably As in the embodiment, the BAC containing the ephrin-A5 enhancer factor is produced by recombining each of the above factors except for the factors caused by ephrin-A5.

Specifically, the step of preparing the recombinant expression vector of (I) is prepared based on the 5 'terminal sequence of (1) the first exon sequence of the ephrin-A5 gene in (I-1) plasmid vector. A homology A cancer sequence comprising the sequence set forth in SEQ ID NO: 2 functioning as a promoter of lean-A5, (b) a reporter gene downstream of the homology A cancer and having a recombinant sequence at both ends thereof and the gene Antibiotics with a 3 'regulatory sequence, (c) an ephrin-A5-Fc gene located underneath the reporter gene and a 3' regulatory sequence, (d) a recombinant sequence located underneath the ephrin-A5-Fc gene, with a recombinant sequence at both ends A homologous B cancer sequence which is constructed based on the resistance gene and (e) the 3 'end sequence of the first exon sequence of the ephrin-A5 gene located below the antibiotic resistance gene and comprising the sequence set forth in SEQ ID NO: 3. Insert target vector (I-2) preparation of the recombinant enzyme gene which induces homologous recombination in the BAC host cell comprising the ephrin-A5 enhancer factor and the ephrin-A5 first exon sequence. Homologous recombination of the target vector into the BAC first exon sequence via homology A and B cancers of the target vector by transformation with an expression vector and by the recombinant enzyme, (I-3) an antibiotic of the target vector Selecting a transformed BAC host cell in which the target vector is resistant to antibiotics by a resistance gene, wherein the target vector is recombined with BAC, and (I-4) isolating BAC recombined with the target vector from the host cell. It is configured by.

In this way, the recombinant BAC is a promoter of ephrin-A5 (homologous cancer A), a reporter gene downstream of the promoter and the recombinant sequence at both ends, a 3 'regulatory sequence of the gene, and a lower part of the reporter gene. And the enhancer of ephrin-A5-Fc gene located at the 3 'regulatory sequence of the gene and ephrin-A5 located below the ephrin-A5-Fc gene. Except for the ephrin-A5 enhancer included in the BAC, the remaining factors are all inserted into the BAC by homologous recombination of the target vector.

Recombinant enzymes that induce homologous recombination of step (I-2) can be used any recombinant enzyme known in the art, in the following examples lambda phage recombinant enzyme was used.

When the antibiotic resistance gene of the recombinant expression vector transforms the BAC host cell into an expression vector containing a recombinant enzyme that specifically recognizes the recombinant sequences at both ends of the antibiotic resistance gene and cleaves and binds the gene between the recombinant sequences. Can be removed. Since the antibiotic resistance gene does not need to be specifically introduced into fertilized eggs or transgenic mice, it is preferable to remove it. As the sequence specific recombinase, Cre recombinase, FLP recombinase, Kw integrase and the like may be used. In the following examples of the present invention, FLP recombinase was used.

On the other hand, after the production of such expression vector is introduced into the mouse to produce a transgenic mouse, the method of producing a transgenic mouse can be used any method known in the art.

As such a method, a method of introducing the expression vector into a fertilized egg and transplanting the fertilized egg into a surrogate mother can be used as in the following examples. In addition to fertilized eggs, it can also be introduced into ES cells with early embryos, multipotents, and the like.

As the introduction method, an electroporation method, a liposome method, a calcium phosphate method, a micro injection method, or the like can be used. After introduction, it can be transplanted into surrogate mothers, such as fertility mice, and developed into embryos and adults.

Specific methods for producing transgenic mice are described in Intl. Rev. Cytol., 115: 171-229 (1989), Clinical and Experimental Pharmacology and Physiology, Supp. 3: S81-S87, 1996, Transgenic Animal Technology, A Handbook, 1994, ed., Carl A. Pinkert, Academic Press, Inc.

As described above, the present invention can provide a transgenic mouse useful for studying the function of ephrin-A5 and a method for producing the transgenic mouse.

1 is a schematic diagram showing which positions of the ephrin-A5 gene are RPCI-23-4O15, 375B7, 211L12, and 23O22, which are BACs.
2 is a schematic diagram of pGEM-11Z, a lacZ expression target vector for use in BAC homologous recombination.
Figure 3 is a schematic diagram of the process of recombination lacZ expression target vector to BAC.
4 is a schematic diagram of the lacZ gene recombined with BAC and X-gal staining photographs of embryos at days 8.5, 9.5 and 10.5 of the mice transformed with the recombinant BAC.
Fig. 5 is a schematic diagram of RPCI-23-375B7 BAC from which + 0.2kb to + 30.5kb region was deleted and X-gal staining photographs of embryos at days 9.5 and 10.5 days of mice transformed with the BAC.
6 to 8 are schematic diagrams of the promoter, lacZ gene and C1 to C3 fragments of the ephrin-A5 gene inserted into the pGEM-11Z vector.
Figure 9 is a schematic of the location of the C1 to C3 fragments and X-gal staining photographs of embryos on day 8.5 and day 9.5 of transgenic mice showing the C1 to C3 fragments induce the expression of lacZ.
10 is a schematic diagram of pGEM-11Z, an eGFP expression target vector for use in BAC homologous recombination.
11 is a schematic diagram of the process of recombination of eGFP expression target vector into BAC.
12 is a fluorescence micrograph of the 10.5 days of embryonic development of mouse eGFP ^floxed -eA5-Fc transformed with BAC 23O22 inserted with an eGFP expression target vector.
Figure 13 shows ^embryonic 9.5, 10.5, 11.5, 12.5, 13.5, 14.5, 14.5 and 16.5 days of embryonic development of eGFP ^floxed -eA5-Fc transgenic mice and eGFP ^floxed -eA5-Fc / Wnt1-Cre transgenic mice. Corresponding brain shape picture.
14 is a cross-sectional photograph of the 12.5 days of embryonic development of eGFP ^floxed -eA5-Fc transgenic mice and eGFP ^floxed -eA5-Fc / Wnt1-Cre transgenic mice.
15 shows TUNEL assay results of eGFP ^floxed -eA5-Fc transgenic mice and eGFP ^floxed -eA5-Fc / Wnt1-Cre transgenic mice.
Figure 16 shows the results of immunohistochemistry of caspase-3 in the active form of eGFP ^floxed -eA5-Fc transgenic mice and eGFP ^floxed -eA5-Fc / Wnt1-Cre transgenic mice.

Hereinafter, the present invention will be described with reference to reference examples and examples. However, the scope of the present invention is not limited to these reference examples and examples.

< Reference Example > Ephrine -A5 Enhancer  Separation of arguments

< Reference Example  1> BAC  Purchase of clones

The genomic sequence of the ephrin-A5 gene, which consists of five exons and is approximately 270 kb (NCBI GenBank ID: 13640), contains a BAC clone containing a region that is expected to have an enhancer of the ephrin-A5 gene. Four clones of RPCI-23-4O15, 375B7, 211L12 and 23O22 clones, 150-250 kb in size, were purchased from the Children's Hopspital Oakland Research Institute.

RPCI-23-4O15 is located from -207.5 kb to +11.6 kb, based on + 1b as the first nucleotide of the first exon of ephrin-A5. RPCI-23-375B7 is located from -154.4kb to + 57.7kb, based on + 1b as the initial nucleotide of the first exon of ephrin-A5. RPCI-23-211L12 is located from -71.5 kb to +115.3 kb, based on + 1b as the initial nucleotide of the first exon of ephrin-A5. RPCI-23-23O22 is located from -6.7 kb to +214.8 kb, based on + 1b as the first nucleotide of the first exon of ephrin-A5.

In FIG. 1, each of the BACs corresponds to which region of the ephrin-A5 gene.

< Reference Example  2> BAC Of target vector capable of homologous recombination

In order to know the enhancer region of the ephrin-A5 gene, a target vector capable of homologous recombination of a reporter gene into four BACs of Reference Example 1 was prepared.

First, a forward primer sequence (SEQ ID NO: 5) and a reverse primer sequence (SEQ ID NO: 6) for cloning homology arm A based on both terminal sequences (adjacency sequences) of the first exon of the ephrin-A5 gene And a forward primer sequence (SEQ ID NO: 7) and a reverse primer sequence (SEQ ID NO: 8) for cloning homology arm B.

Next, arm A was the first exon of the ephrin-A5 gene as a template, PCR amplification using SEQ ID NOs: 5 and 6 to obtain a fragment, and both ends of the fragment were cut with SfiI / SacI, pGEM-11Z. Vector (Promega) was inserted at the site cut with SfiI / SacI.

LacZ containing SV40 polyadenylation signal sequence (LacZ-pA) and kanamycin resistant cassettes with FRT sites at both ends were digested with XhoI / NotI ( Journal). of Biochemistry and Molecular Biology , Vol. 40, No. 5, 2007, pp. 656-661), A arm was inserted into the site cut with XhoI / NotI of the inserted vector.

Subsequently, the B arm was used as the template for the first exon of the ephrin-A5 gene, and PCR amplification using SEQ ID NOs: 7 and 8 to obtain a fragment, and both ends of the fragment were cut with NotI / NsiI, and lacZ- pA, Kanamycin-resistant cassette and A-arm were inserted into the NotI / NsiI cleavage site of the inserted vector.

FIG. 2 is a schematic diagram showing the insertion of the kanamycin resistance cassette having the A- and B-arms, the lacZ-containing SV40 polyadenylation signal sequence, and the FRT site at both ends thereof, on the pGEM-11Z.

The finished vector was then cleaved with SfiI / NsiI and purified on 0.8% agarose gel and used for the next experiment.

< Reference Example  3> BAC Homologous recombination

Reference Example 3-1 First Recombination

BAC host cells resistant to chloramphenicol antibiotics were streaked on agar plates containing chloramphenicol (Cam) antibiotics and grown overnight at 37 ° C. . Single colonies were selected from the grown colonies and placed in 5 ml Luria broth (LB) containing Cam and grown overnight at 37 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.7-0.8. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid layer was centrifuged again to separate the liquid layer and the cell layer, and then the liquid was discarded and dissolved again in ice-cold water. This process was repeated one more time. After dissolving the obtained cell pellet with 50 μl of ice-cold water, ampicillin (Amp) with lambda phage recombinase induced expression by arabinose PKOBEGA plasmid with resistance gene ( Nature , Vol. 435, 2005, pp. 1244-1250) (INSTITUT PASTEUR) was added and mixed well, and transformed by electroporation method. After addition of 1 ml of LB, the cells were incubated at 30 ° C. for 1 hour, and spread overnight at 30 ° C. by spreading on an agar plate to which Cam and ampicillin (Amp) were added. The next day, Cam and Amp-resistant single colonies were selected and grown in 37 ml Luria broth (LB) containing Cam and Amp overnight at 37 ° C. The next day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.05-0.1. 10% L (+) arabinose (arabinose, Sigma A-3256) was treated in a culture solution at a final concentration of 0.2% and grown at 30 ° C for 1 hour. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid was centrifuged again to separate the liquid layer and the cell layer, and then the liquid was discarded and again dissolved in ice-cold water. This process was repeated one more time. The obtained cell pellet was dissolved in 50 µl of ice-cold water, and then mixed with 1 µg of the cleaved target vector obtained in Reference Example 2 and mixed well, followed by transformation by electroporation. . After addition of 1 ml of LB, the cells were incubated at 37 ° C. for 1 hour, and spread overnight at 37 ° C. by spreading on agar plates to which Cam and kanamycin (Kan) were added.

Referring to FIG. 3, it can be seen that the target vector of Reference Example 2 is introduced into a host cell containing BAC and pKOBEGA plasmid and recombined with BAC (FIGS. 2A and 2B).

Reference Example 3-2 Second Recombination

Among the first recombinant BAC clones selected for antibiotic resistance, single colonies were selected and grown in 37 ml Luria broth (LB) containing Cam and Kan overnight at 37 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.7-0.8. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid was centrifuged again to separate the liquid and cell layers, discarded the liquid, and dissolved in ice-cold water again. This process was repeated one more time. After dissolving the obtained cell pellet with 50 μl of ice-cold water, the FLP plasmid containing the resistance gene of tetracycline (Tet), which can induce Flipase expression, which induces FRT-dependent homologous recombination ( Gene Bridges) 10ng was mixed well, and transformed by electroporation. After addition of 1 ml of LB, the cells were incubated at 30 ° C. for 1 hour and spread overnight on an agar plate containing Cam, Kan and tetracycline (Tet) at 30 ° C. overnight. Grown. The next day, Cam, Kan and Tet-resistant single colonies were selected and grown in 5 ml Luria broth (LB) containing Cam overnight at 30 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 20 ml of LB and grown to OD ₆₀₀ = 0.5 for 2 hours. 10 ml of this was transferred again and incubated for 15 minutes in a 42 degreeC waterbath. Quickly transferred to a bath of ice, the temperature was drastically lowered and left for 5 minutes. 10-100 μl of these cells were spread on agar plat to which Cam was added and grown overnight at 37 ° C. The FLP plasmid expresses Flipase, which homologously removes the site between two FRTs at 35-42 ° C. At 37 ° C, it does not replicate and is diluted and disappeared. Therefore, the second recombination removed a kanamycin-resistant portion between the two FRTs, and a single colony was selected by temperature screening to remove the tetracycline-resistant portion.

3 shows a schematic diagram of a state in which the lacZ gene is inserted in the BAC by the second recombination (see (C) of FIG. 2).

Reference Example 4 Construction of Transgenic Mice

Reference Example 4-1 Purification of Recombinant DNA

Recombinant BAC DNA was purified using a large-construct kit (Qiagen).

Check the quality and quantity of DNA on the gel, dilute BAC DNA in injection buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA pH 8.0 and 100 mM NaCl) at a concentration of 10 ng / μl of the 2X mix the polyamine-based 48 hours was injected into the male pronucleus of the fertilized egg fine (Nat Rev Neurosci, vol 2, 2001, pp 861-870;........ Dev Dyn, vol 226, 2003, pp. 596-603; Genesis , vol. 38, 2004, pp. 39-50).

Reference Example 4-2 Sampling of Embryos

As a mouse to collect fertilized eggs, 3 to 4 week old females of C57L / 6 species were used. First, 0.1 ml of PMSG (pregnant mare serum gonadotropin; 50 IU / ml) was injected into the abdominal cavity of 3-4 weeks of mice with a time difference to induce hyperovulation of C57BL / 6 female. After 46 hours, 2.5 units of HCG (human chronic gonadotropin (25 IU / ml) were injected intraperitoneally and then crossed with C57BL / 6 male mice. The next day, the oviduct of the mouse with the vaginal plug was removed, and the ampula was injured with a needle. Washed hyaluronidase components. Only 1-cell fertilized eggs were recovered and transferred to M16 medium to stabilize the fertilized eggs in a CO ₂ incubator for 4 hours.

<Reference Example 4-3> Fine injection (microinjection) of the recombinant DNA of the embryos

Recombinant DNA was injected into the male pronucleus of the male pronucleus and male pronucleus of the fertilized eggs collected by microinjection method using high magnification microscope. Fertilized eggs were cultured over-night in a CO ₂ incubator.

REFERENCE EXAMPLE 4-4 Transplanting Embryos into Surrogate Mother Tubes

The day before, only 2-cell differentiated embryos were selected from fertilized eggs injected with recombinant DNA, and transplanted into prepared oviducts of ICR female surrogate mothers. After anesthesia, ICR female was anesthetized, the hair at the center of the back was removed, and the skin near the kidney and the muscle at the site were incised. After ovaries, fallopian tubes and parts of the uterus were taken out of the body, the ovarian follicles covering the ovaries were torn and exposed to the oocytes, and the fertilized eggs (about 15 to 25) were inserted into the pipettes for implantation and slowly injected by inserting them into the oocytes. . After transplantation, the ovaries, fallopian tubes and uterus were put back into the abdominal cavity and sutured.

< Reference Example  5> Embryo X- gal  dyeing

Female mice pregnant at the desired embryonic stage were dissected to obtain embryos corresponding to days 8.5, 9.5 and 10.5 of development. Subsequently, the uterine membrane, yolk sac and amnion sac surrounding the embryo were removed. The isolated embryos were then washed once with 1 × PBS (phosphate buffered saline) and then incubated for 10 minutes in a fixing solution. Subsequently, it was washed three times for 10 minutes with a washing solution. Subsequently, a staining solution containing X-gal was added thereto and then incubated at 37 ° C. for 5 hours.

The staining photograph of the embryo is shown in FIG. 4. 4A is a schematic diagram of a state in which the lacZ gene is inserted into BAC by two recombination of <Reference Example 3>, and FIG. 4B is X-gal of a mouse embryo transformed with BAC into which the lacZ gene is inserted. It is a dyed picture.

Referring to FIG. 4, the embryo transformed with lacZ-containing RPCI-23-4O15 did not stain the hepatic and midbrain center lines, whereas the embryo transformed with other BACs, particularly lacZ-containing RPCI-23-375B7, showed hepatic and midbrain. You can see that the centerline is dyed.

The results of FIG. 4 show that the enhancer of ephrin-A5 exists between + 11.6kb and + 57.7kb.

< Reference Example  6> RPCI -23-375B7 BAC ( BAC ) 375B7, a target vector capable of homologous recombination del30 .3 manufacturing

Based on the initial nucleotide of the first exon of ephrin-A5 in the RPCI-23-375B7 BAC region as + 1b, a target vector capable of deleting 30.3 kb sizes from +0.2 kb to +30.5 kb was prepared. Homology A arm was PCR amplified with a forward primer (SEQ ID NO: 9) and a 5'-reverse primer (SEQ ID NO: 10) prepared based on both terminal sequences of the + 0.2kb to + 30.5kb region to obtain fragments. Both ends of the fragment were cut with SfiI / SacI, and the pGEM-11Z vector was inserted into the site cut with SfiI / SacI. The LacZ containing SV40 polyadenylation signal sequence (lacZ-pA) and the kanamycin resistant cassette with FRT sites at both ends were cut with XhoI / NotI ( Journal). of Biochemistry and Molecular Biology , Vol. 40, No. 5, 2007, pp. 656-661), A arm was inserted into the site cut with XhoI / NotI of the inserted vector. Subsequently, the B arm was similarly PCR amplified with a forward primer (SEQ ID NO: 11) and a reverse primer (SEQ ID NO: 12) prepared based on both terminal sequences of the + 0.2kb to + 30.5kb region to obtain fragments. Both ends of were cut with NotI / NsiI and inserted into a site cut with NotI / NsiI of a vector in which a lacZ-pA, Kan-resistant cassette and A arm were inserted. Subsequently, the completed vector was cleaved with SfiI / NsiI and purified on 0.8% agarose gel, which was then recombined into BAC (BAC) clone in the same manner as in <Reference Example 3>, and <Reference Example 4 > In the same manner as the injection into the fertilized egg of the mouse was produced a transformed mouse. Subsequently, X-gal staining was performed in the same manner as in <Reference Example 5>.

The results are shown in Fig. Referring to Figure 5 it can be seen that both the brain and the dorsal centerline of the embryos corresponding to days 9.5 and 10.5 days of development did not stain.

Combining the results of FIG. 5 and the results of FIG. 4 of Reference Example 5, it is suggested that an enhancer exists at a region between + 11.6kb and + 30.5kb.

Based on these results, in order to confirm the correct position of the enhancer, each fragment was divided into three fragments (C1, C2, and C3) having a size of about 5 to 7 kb in the section of 11.6 kb to +30.5 kb in the following experiment. Was prepared a recombinant vector bound to the lacZ gene, and a transgenic mouse was prepared with this to determine which fragment has the function of an enhancer.

< Reference Example  7> Enhancer  Experiment to confirm

REFERENCE EXAMPLE 7-1 Preparation of a Recombinant Vector Containing C1 , C2, and C3 Fragments

Reference Example 7-1-1 Preparation of Recombinant Vector Containing C1 Fragment

PCR (polymerase chain reaction) amplification of the ephrin-A5 promoter (see SEQ ID NO: 2) with a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) to obtain fragments, and the ends of the fragments were identified as SfiI / SacI. The pGEM-11Z vector was inserted into the site cut with SfiI / SacI. Subsequently, the vector into which the ephrin-A5 promoter was inserted was cleaved with XbaI, and a linker was made with a site capable of cleavage with SpeI. Subsequently, the promoter was inserted and the linker cut vector was cut with XhoI, and then lacZ containing SV40 polyadenylation signal sequence was inserted into the site. Subsequently, the PCR-A5 C1 fragment was PCR amplified with a forward primer (SEQ ID NO: 13) and a reverse primer (SEQ ID NO: 14) to obtain a fragment, and both ends of the fragment were cleaved with NheI / NotI, and the ephrin-A5 promoter was The lacZ was inserted and the linker was inserted into the site cut with SpeI / NotI (NheI and SpeI have compatible ends that can be ligationed together). Subsequently, the finished vector was cut with SfiI / NotI and purified on 0.8% agarose gel, which was used for microinjection into fertilized eggs of mice.

Figure 6 shows a schematic of the insertion of the ephrin-A5 promoter, lacZ gene and C1 fragment into the pGEM-11Z vector.

REFERENCE EXAMPLE 7-1-2 Preparation of a Recombinant Vector Containing a C2 Fragment, Construction of a Transgenic Mouse, and X- gal Staining

PCR (polymerase chain reaction) amplification of the ephrin-A5 promoter with a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) to obtain a fragment, and both ends of the fragment were cut with SfiI / SacI, pGEM-11Z The vector was inserted at the site cut with SfiI / SacI. Subsequently, the vector into which the ephrin-A5 promoter was inserted was cleaved with XbaI, and a linker was made with a site capable of cleavage with SpeI. Subsequently, the promoter was inserted and the linker cut vector was cut with XhoI, and then lacZ containing SV40 polyadenylation signal sequence was inserted into the site. Subsequently, the PCR-A5 C2 fragment was PCR amplified with a forward primer (SEQ ID NO: 15) and a reverse primer (SEQ ID NO: 16) to obtain a fragment, and both ends of the fragment were cleaved with NheI / NotI, and the ephrin-A5 promoter was The lacZ was inserted and the linker-linked vector was inserted into the site cut with SpeI / NotI. Subsequently, the finished vector was cut with SfiI / NotI and purified on 0.8% agarose gel, which was used for microinjection into fertilized eggs of mice.

Figure 7 shows a schematic of the ephrin-A5 promoter, lacZ gene and C2 fragment inserted into the pGEM-11Z vector.

REFERENCE EXAMPLE 7-1-3 Preparation of Recombinant Vector Containing C3 Fragment, Construction of Transgenic Mice, and X- gal Staining

PCR (polymerase chain reaction) amplification of the ephrin-A5 promoter with a forward primer (SEQ ID NO: 5) and a reverse primer (SEQ ID NO: 6) to obtain a fragment, and both ends of the fragment were cut with SfiI / SacI, pGEM-11Z The vector was inserted at the site cut with SfiI / SacI. Subsequently, the vector into which the ephrin-A5 promoter was inserted was cleaved with XbaI, and a linker was made with a site capable of cleavage with SpeI. Subsequently, the promoter was inserted and the linker cut vector was cut with XhoI, and then the lacZ containing SV40 polyadenylation signal sequence was inserted into the site. Subsequently, the PCR-A5 C3 fragment was PCR amplified with a forward primer (SEQ ID NO: 17) and a reverse primer (SEQ ID NO: 18) to obtain a fragment, and both ends of the fragment were cleaved with NheI / NotI. The lacZ was inserted and the linker-linked vector was inserted into the site cut with SpeI / NotI. Subsequently, the finished vector was cut with SfiI / NotI and purified on 0.8% agarose gel, which was used for microinjection into fertilized eggs of mice.

Figure 8 shows a schematic of the insertion of the ephrin-A5 promoter, lacZ gene and C2 fragment into the pGEM-11Z vector.

Reference Example 7-2 Transgenic Mouse Preparation and X- gal Staining

Each recombinant vector obtained in <Reference Example 6-1> was microinjected into the fertilized egg of a mouse in the same manner as in <Reference Example 4> to prepare a transgenic mouse, and then X- gal staining was performed.

The staining results are shown in FIG. 9. FIG. 9A is a schematic diagram of the positions of C1, C2 and C3 fragments, and FIG. 9B is X-gal staining of embryos at day 9.5 of development of the transgenic mice showing whether the three fragments induce the expression of lacZ. It is a photograph. Referring to B of FIG. 9, it can be seen that the C3 fragment is an enhancer. The sequence of the C3 fragment is disclosed in SEQ ID NO: 1.

< Example > Ephrine Construction of Transgenic Mice Useful for Functional Study of A-A5 Gene

< Example  1> Ephrine -A5 Enhancer  Containing arguments BAC Target vector homologous to targeting vector Manufacturing

The pGEM11Z vector (Promega) was cut with XbaI, and Kan-resistant cassettes with FRT sites at both ends were cut with SpeI (XbaI and SpeI have compatible cohesive ends, which allows for the ligation of the cut sites. ).

Then, a forward primer sequence (SEQ ID NO: 5) and a reverse primer sequence (SEQ ID NO: 6) for cloning homology arm A based on both terminal sequences of the first exon of the ephrin-A5 gene were synthesized, In addition, a forward primer sequence (SEQ ID NO: 7) and a reverse primer sequence (SEQ ID NO: 8) for cloning homology arm B were synthesized.

Next, arm A was the first exon of the ephrin-A5 gene as a template, PCR amplification using primers of SEQ ID NOs: 5 and 6 to obtain fragments, and both ends of the fragments were cut with SfiI / SacI, and kanamycin. A pGEM-11Z vector (Promega) in which a Kan-resistant cassette was inserted was inserted into a site cut with SfiI / SacI.

In addition, the arm B was PCR amplified using the primers of SEQ ID NOs: 7 and 8 to obtain a fragment, and both ends of the fragment were cut with NotI / NsiI, and the kanamycin resistant cassette and the arm A were armed. ) Was inserted into the site cut with NotI / NsiI.

The homology A cancer sequence and the homology B cancer sequence can be found in SEQ ID NO: 2 and SEQ ID NO: 3, respectively.

Subsequently, NdeI can be cleaved by attaching a linker to the site where SfiI can be cleaved in the front part of the A arm and the site where the NsiI can be cleaved in the rear part of the B arm. To site.

Subsequently, the SV40 polyadenylation signal sequence containing the ephrin-A5-Fc gene (SEQ ID NO: 4) was cleaved with SalI to insert a kanamycin resistant cassette, an A arm and a B arm. Inserted into the site cut with SalI of the vector. Subsequently, the eGFP-SV40 polyadenylation signal sequence cassette with loxP sites at both ends was then replaced with ephrin-A5-Fc containing SV40 polyadenylation signal sequence, kanamycin resistant cassette, A arm. And B arm were inserted into the site cut with SacI of the inserted vector.

A schematic diagram of the target vector thus produced is shown in FIG. 10.

The completed target vector was then cleaved with NdeI and purified on 0.8% agarose gel and recombined into BAC clones.

< Example  2> BAC  Homologous recombination ( BAC homologous recombination )

Example 2-1 First Recombination

RP23-23O22 BAC host cells resistant to chloramphenicol antibiotics were streaked on agar plates containing chloramphenicol Cam antibiotics overnight at 37 ° C. ) Grown. Single colonies were selected from the grown colonies and placed in 5 ml Luria broth (LB) containing Cam and grown overnight at 37 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.7-0.8. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid layer was centrifuged again to separate the liquid layer and the cell layer, and then the liquid was discarded and dissolved again in ice-cold water. This process was repeated one more time. After dissolving the obtained cell pellet with 50 µl of ice-cold water, ampicillin (Amp) with a lambda phage recombinase induced expression by arabinose PKOBEGA plasmid with resistance gene ( Nature , Vol. 435, 2005, pp. 1244-1250) (INSTITUT PASTEUR) was added and mixed well, and transformed by electroporation method. After addition of 1 ml of LB, the cells were incubated at 30 ° C. for 1 hour, and spread overnight at 30 ° C. by spreading on an agar plate to which Cam and ampicillin (Amp) were added. The next day, Cam and Amp-resistant single colonies were selected and grown in 37 ml Luria broth (LB) containing Cam and Amp overnight at 37 ° C. The next day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.05-0.1. 10% L (+) arabinose (arabinose, Sigma A-3256) was treated in a culture solution at a final concentration of 0.2% and grown at 30 ° C for 1 hour. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid was centrifuged again to separate the liquid layer and the cell layer, and then the liquid was discarded and again dissolved in ice-cold water. This process was repeated one more time. The obtained cell pellet was dissolved in 50 µl of ice-cold water, and then mixed with 1 µg of the cleaved target vector obtained in Example 2 and mixed well, followed by transformation by electroporation. . After addition of 1 ml of LB, the cells were incubated at 37 ° C. for 1 hour, and spread overnight at 37 ° C. by spreading on agar plates to which Cam and kanamycin (Kan) were added.

11 shows a schematic diagram of the target vector of <Example 1> introduced into a host cell containing BAC and pKOBEGA plasmid and recombined into BAC (FIGS. 11A and 11B).

Example 3-2 Second Recombination

Among the first recombinant BAC clones selected for antibiotic resistance, single colonies were selected and grown in 37 ml Luria broth (LB) containing Cam and Kan overnight at 37 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 10 ml LB and grown to OD ₆₀₀ = 0.7-0.8. The grown cultures were collected in 1.5 ml microtubes, centrifuged to obtain pellets, and then dissolved in 888 µl of ice-cold water. The liquid was centrifuged again to separate the liquid and cell layers, discarded the liquid, and dissolved in ice-cold water again. This process was repeated one more time. After dissolving the obtained cell pellet with 50 μl of ice-cold water, the FLP plasmid containing the resistance gene of tetracycline (Tet), which can induce Flipase expression, which induces FRT-dependent homologous recombination ( Gene Bridges) 10ng was mixed well, and transformed by electroporation. After addition of 1 ml of LB, the cells were incubated at 30 ° C. for 1 hour and spread overnight on an agar plate containing Cam, Kan and tetracycline (Tet) at 30 ° C. overnight. Grown. The next day, Cam, Kan and Tet-resistant single colonies were selected and grown in 5 ml Luria broth (LB) containing Cam overnight at 30 ° C. The following day, 1 ml (OD ₆₀₀ = 1.2) of the culture grown on the previous day was transferred to 20 ml of LB and grown to OD ₆₀₀ = 0.5 for 2 hours. 10 ml of this was transferred again and incubated for 15 minutes in a 42 degreeC waterbath. Quickly transferred to a bath of ice, the temperature was drastically lowered and left for 5 minutes. 10-100 μl of these cells were spread on agar plat to which Cam was added and grown overnight at 37 ° C. The FLP plasmid expresses Flipase, which homologously removes the site between two FRTs at 35-42 ° C. At 37 ° C, it does not replicate and is diluted and disappeared. Therefore, the second recombination removed a kanamycin-resistant portion between the two FRTs, and a single colony was selected by temperature screening to remove the tetracycline-resistant portion.

11 shows a schematic diagram of the kanamycin resistance gene removed from the BAC by the second recombination (see (C) of FIG. 11).

< Example  3> Construction of Transgenic Mice

Example 3-1 Purification of Recombinant DNA

Recombinant BAC DNA was purified using a large-construct kit (Qiagen).

Check the quality and quantity of DNA on the gel, dilute BAC DNA in injection buffer (10 mM Tris-HCl pH 7.5, 0.1 mM EDTA pH 8.0 and 100 mM NaCl) at a concentration of 10 ng / μl of the 2X mix the polyamine-based 48 hours was injected into the male pronucleus of the fertilized egg fine (Nat Rev Neurosci, vol 2, 2001, pp 861-870;........ Dev Dyn, vol 226, 2003, pp. 596-603; Genesis , vol. 38, 2004, pp. 39-50).

<Example 3-2> Embryo Collection

As a mouse to collect fertilized eggs, 3 to 4 week old females of C57L / 6 species were used. First, 0.1 ml of PMSG (pregnant mare serum gonadotropin; 50 IU / ml) was injected into the abdominal cavity of 3-4 weeks of mice with a time difference to induce hyperovulation of C57BL / 6 female. After 46 hours, 2.5 units of HCG (human chronic gonadotropin (25 IU / ml) were injected intraperitoneally and then crossed with C57BL / 6 male mice. The next day, the oviduct of the mouse with the vaginal plug was removed, and the ampula was injured with a needle. Washed hyaluronidase components. Only 1-cell fertilized eggs were recovered and transferred to M16 medium to stabilize the fertilized eggs in a CO ₂ incubator for 4 hours.

<Example 3-3> Fine injection (microinjection) of the embryos of the recombinant DNA

Recombinant DNA was injected into the male pronucleus of the male pronucleus and male pronucleus of the fertilized eggs collected by microinjection method using high magnification microscope. Fertilized eggs were cultured over-night in a CO ₂ incubator.

Example 3-4 Embryo Transfer to Surrogate Mother Oviduct

The day before, only 2-cell differentiated embryos were selected from fertilized eggs injected with recombinant DNA, and transplanted into prepared oviducts of ICR female surrogate mothers. After anesthesia, ICR female was anesthetized, the hair at the center of the back was removed, and the skin near the kidney and the muscle at the site were incised. After ovaries, fallopian tubes and parts of the uterus were taken out of the body, the ovarian follicles covering the ovaries were torn and exposed to the oocytes, and the fertilized eggs (about 15 to 25) were inserted into the pipettes for implantation and slowly injected by inserting them into the oocytes. . After transplantation, the ovaries, fallopian tubes and uterus were put back into the abdominal cavity and sutured.

< Example  4> Observation by fluorescence microscope

Female mice pregnant at the desired embryonic stage were dissected to obtain embryos corresponding to day 10.5 of development. Subsequently, the uterine membrane, yolk sac and amnion sac surrounding the embryo were removed. The isolated embryos were then washed once with 1 × PBS (phosphate buffered saline) and then incubated for 10 minutes in a fixing solution. Then, washed three times with a washing solution (washing solution) three times, and observed by fluorescence microscope (Olympus).

A fluorescence microscope photograph is shown in FIG. 12. Referring to FIG. 12, ephrin-A5 is expressed at 10.5 days of embryonic development by expression of mouse eGFP ^floxed- eA5-Fc eGFP transformed with BAC 23O22 with a target vector. It can be seen that fluorescence is expressed in the center line of the middle brain (mesencephalon) and the liver (diencephalon).

< Example  5> eGFP ^floxed - eA5 - Fc Of Wnt1 - Cre  Transgenic mouse

The transgenic mouse (eGFP ^floxed -eA5-Fc) obtained in <Example 4> was crossed with the Wnt1-Cre transgenic mouse (The Jackson Laboratory) to ^prepare an eGFP ^floxed -eA5-Fc / Wnt1-Cre transgenic mouse. Embryos were separated and washed on the 9.5 days, 10.5 days, 11.5 days, 12.5 days, 13.5 days, 13.5 days, 14.5 days and 16.5 days in the same manner as in <Example 4> and the brain shape was observed.

The results are shown in column B of FIG. 13. Column A in Figure 13 is a photograph corresponding to 9.5 days, 10.5 days, 11.5 days, 11.5 days, 12.5 days, 13.5 days, 14.5 days and 16.5 days of embryo development of the eGFP ^floxed -eA5-Fc transgenic mice obtained in Example 4 to be. Comparing the photo of column B with the photo of column A, the brain size decreases from day 9.5 of embryonic development, and it can be seen that the two facial faces are not properly developed. This can be expected because the eGFP moiety is removed by Cre and the ephrinA5-Fc protein following it is expressed and binds to the Eph receptor, thereby eventually blocking the binding of Ephrine-A5 to the Eph receptor.

< Example  6> Brain cross-section and TUNEL assay

Example 6-1 Brain Sectional Scan

^Embryo development 12.5 days old eGFP ^floxed- eA5-Fc / Wnt1-Cre transgenic mouse embryos of Example 5 were prepared as paraffin-embedded tissue fragments. The tissue fragments were immersed in different concentration series of xylene and ethanol solutions, subjected to dewaxing and rehydration, followed by cresyl violet staining, mounting with canada balsam, and comparison of their shape differences with normal mice. It was.

The results are shown in B of FIG. 14, and when compared with eGFP ^floxed -eA5-Fc transgenic mice (A of FIG. 14), the ventricles of hepatic and midbrain were smaller than those of normal mice. FIG. 14A is a cross-sectional photograph of the embryonic day 12.5 days of the eGFP ^floxed- eA5-Fc transgenic mouse obtained in Example 4. FIG.

<Example 6-2> TUNEL assay

The eGFP ^floxed- eA5-Fc / Wnt1-Cre transgenic mouse embryos obtained in Example 5 were obtained and prepared as paraffin-embedded tissue fragments. The tissue fragments were immersed in different concentration series of ethanol and xylene solutions for dewaxing and rehydration. Subsequently, the proteinase K solution (10 μg / ml in 10 mM Tris / HCl, pH 7.4-8) was treated at 37 ° C. for 15 minutes. Subsequently, using an In situ cell death detection kit, fluorescein (Roche), the Enzyme solution and the Label solution were mixed at a ratio of 1: 9, respectively, and treated at 37 ° C. for 60 minutes. After washing with PBS, photographed using a fluorescence microscope and the TUNEL positive cell was counted and the results are shown in a graph with a photograph in FIG.

Referring to FIG. 15, it can be observed that transgenic mice have about 10 times more cell death than eGFP ^floxed -eA5-Fc transgenic mice (A of FIG. 15) in the ^hepatic and midbrain regions. 15A is a cross-sectional photograph of the brain of the eGFP ^floxed -eA5-Fc transgenic mouse obtained in Example 4. a, b and c are cut planes.

< Example  7> Immunohistochemical tests ( immunohistochemistry assay )

Since apoptosis is known to be involved in caspase-3 at the end of the signal transduction process (Li and Yuan, Oncogene 27: 6194-206, 2008), eGFP ^floxed -eA5-Fc / Wnt1-Cre obtained in Example 5 above. To determine how much of the active form of caspase-3 is expressed in transgenic mice, an antibody specific for the active form of caspase-3 (Cleaved Caspase-3 (Asp175) Antibody: Preparation of Cell Signaling: 9661) was used for immunohistochemical examination.

Specifically, embryos of 10.5 days of development were obtained, placed in 4% PFA (fixed solution), fixed at 4 ° C over night, and washed with 1X PBS. Xylene and paraffin were embedded sequentially, and the slices were cut in the direction of Saggital and attached to the slide glass. The tissue fragments were immersed in xylene solution of various concentration series for dewaxing and rehydration. Then Antigen retrieval process, 0.01M citrate in DW (pH 6.0) was boiled in a microwave oven for 5 minutes in advance, the tissue was added and boiled for 15 minutes, and then put in ice and cooled for 20 minutes. Then washed with 1X PBS and treated with blocking buffer (5% BSA, 0.5% Tween-20, 5% goat (or alternative) serum in PBS) to the tissue, and reacted at room temperature for 1 hour. Caspase3 antibody was mixed in the blocking buffer, treated to the tissue, reacted at 4 ° C. overnight, and washed with 1x PBS. Fluorescent secondary antibody was mixed with blocking buffer, treated with tissue, reacted at room temperature for 2 hours, washed with 1x PBS, and then mounted with VECTASHIELD Mounting Medium (vector, H1000).

Eotneunde shows the results with the enlarged picture in Figure 16, the active form of caspase-3 is eGFP ^floxed -eA5-Fc transgenic mouse from Conversion (A in FIG. 16) than the ^{eGFP floxed -eA5-Fc / Wnt1-} Cre transgenic mice ( In Figure 16 B) it was confirmed that more expression. 16A is a photograph of the eGFP ^floxed -eA5-Fc transgenic mouse obtained in Example 4.

Attach an electronic file to a sequence list

Claims (13)

(a) a promoter of ephrin-A5,
(b) a reporter gene located at both ends of the promoter and a loxP sequence, a recombinant sequence recognized specifically by the Cre recombinase of bacteriophage P1 , and the 3 'regulatory sequence of the gene,
(c) the ephrin-A5-Fc gene located below the reporter gene and the 3 'regulatory sequence of the gene, and
(d) includes an enhancer of ephrin-A5 located downstream of the ephrin-A5-Fc gene,
The promoter of Ephrin-A5 consists of the sequence set forth in SEQ ID NO: 2,
The ephrin-A5-Fc gene consists of the sequence set forth in SEQ ID NO: 4,
The enhancer factor Ephrin-A5 is a recombinant expression vector, characterized in that consisting of the sequence shown in SEQ ID NO: 1.
The method of claim 1,
The reporter gene is a recombinant expression vector comprising a gene selected from the group consisting of a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, a luciferase gene, a GUS gene, a lacZ gene and a chloramphenicol acetyltransferase gene. .
delete delete The transgenic mouse into which the recombinant expression vector of Claim 1 or 2 was introduced.
(I) A reporter gene having a loxP sequence at both ends and a 3 'regulatory sequence thereof, a recombination sequence specifically recognized by a promoter of ephrin-A5 and Cre recombinase of bacteriophage P1 located downstream of the promoter. A recombinant expression vector comprising an ephrin-A5-Fc gene located below the reporter gene and a 3 'regulatory sequence of the gene, and an enhancer of ephrin-A5 located below the ephrin-A5-Fc gene; And (II) introducing the recombinant expression vector into a mouse which is a host organism.
The promoter of Ephrin-A5 consists of the sequence set forth in SEQ ID NO: 2,
The ephrin-A5-Fc gene consists of the sequence set forth in SEQ ID NO: 4,
The method of manufacturing a transgenic mouse, characterized in that the enhancer factor of ephrin-A5 is composed of the sequence set forth in SEQ ID NO: 1.
The method according to claim 6,
Preparation step of the recombinant expression vector of (I),
(I-1) A plasmid vector comprising (a) the sequence set forth in SEQ ID NO: 2 constructed based on the 5 'end sequence of the first exon sequence of the Ephrin-A5 gene and functioning as a promoter of Ephrin-A5 Homologous A cancer sequence, (b) a reporter gene having a loxP sequence at both ends and a 3 'of the gene, a recombinant sequence located downstream of the homology A cancer and specifically recognized by Cre recombinase of bacteriophage P1 A regulatory sequence, (c) an ephrin-A5-Fc gene and a 3 'regulatory sequence located underneath the reporter gene, and (d) a Saccharomyces cerevisiae derived from the yeast at both ends and located below the ephrin-A5-Fc gene. An antibiotic resistance gene with an FRT recombination sequence specifically recognized by the Flp recombinase, and (e) is located below the antibiotic resistance gene and based on the 3 'end sequence of the first exon sequence of the ephrin-A5 gene. And a step of inserting a homo Lodge arm B sequence consists of the sequence set forth in SEQ ID NO: 3, making the target vector,
(I-2) The produced target vector is transfected into an BAC host cell containing an ephrin-A5 enhancer factor and comprising an ephrin-A5 first exon sequence together with an expression vector of a recombinant enzyme gene that induces homologous recombination. Homologous recombination of the target vector to the BAC first exon sequence via the homology A and B cancers of the target vector by the recombination enzyme;
(I-3) selecting a transformed BAC host cell in which the target vector is recombinant to BAC, which is resistant to antibiotics by an antibiotic resistance gene of the target vector, and
(I-4) A method for producing a transgenic mouse, comprising the step of isolating the recombinant BAC from the target vector in the host cell.
The method of claim 7, wherein
The recombinant enzyme for inducing homologous recombination of step (I-2) is a method for producing a transgenic mouse, characterized in that lambda phage recombinant enzyme.
The method of claim 7, wherein
After the step (I-3), the BAC host cell is transformed into an expression vector comprising a recombinant enzyme that specifically recognizes the recombinant sequence at both ends of the antibiotic resistance gene and cleaves and binds the antibiotic gene between the recombinant sequences. To remove the antibiotic resistance gene of the recombinant expression vector.
The method according to claim 6,
The reporter gene is a transgenic mouse, characterized in that the gene selected from the group consisting of a green fluorescent protein (GFP) gene, a red fluorescent protein (RFP) gene, luciferase gene, GUS gene, lacZ gene and chloramphenicol acetyltransferase gene Method of preparation.
delete delete The method according to claim 6,
The step (II) is a method for producing a transgenic mouse, comprising the step of introducing the expression vector of the (I) step into the fertilized egg and transplanted into the surrogate mother.

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