KR100863241B1 - Transgenic mice overexpressing ?ig-h3 and method for producing thereof - Google Patents

Abstract

The present invention relates to a transgenic mouse overexpressing βig-h3 and a method for producing the same. More specifically, the present invention relates to a transgenic mouse and a method for producing the ocular disease caused by overexpressing βig-h3 specifically in the liver and hypersecreting it into the blood.

The transgenic mice of the present invention can be usefully used for the study of eye development and can be very useful as a therapeutic model for eye diseases such as corneal dystrophy, corneal haze and anterior eye hypoplasia.

βig-h3 protein, transformation, eye disease

Description

Transgenic mice overexpressing βig-h3 and method for producing et al.

Figure 1 shows the manufacturing process of the vector used for the production of βig-h3 overexpressing transgenic mice in the present invention.

Alb E / P: Albumin Enhancer / Promoter

IRES-LacZ-mpl / pA: IRES-LacZ-mP1 intron / poly A

Figure 2 is a photograph confirmed by PCR that the gene construct of the present invention was transplanted to the chromosome of the transgenic mouse according to the present invention.

M: DNA molecular weight marker, F: aid mouse

wt: wild type mouse, tg: transformed mouse

Figure 3 is a photograph confirming the hβig-h3 protein expressed in liver tissue of transgenic mice according to the invention by Western blot.

WT: wild-type mouse (2 months old)

TG1: transgenic mice (2 months old)

TG9: Transgenic Mice (14 Months Old)

TG4: transgenic mouse (12 months old)

Figure 4 is the result of confirming the hβig-h3 protein expressed in the plasma of the transformed mouse according to the present invention by enzyme-linked immunosorbent adsorption (ELISA).

wt: wild type mouse, tg: transgenic mouse

5 is a result of visual observation of the corneal haze of the transgenic mouse according to the present invention.

A: wild-type mouse (2 months old), B: transgenic mouse (2 months old)

C: transgenic mice (14 months old), D: transgenic mice (12 months old)

6 shows the results of histological observation of the eyes of 2 month-old transgenic mice with corneal haze.

A1, A2: wild type mice, B1, B2: transgenic mice

A1, A2, B1, B2: Hematoxylin-eosin staining

A2, B2: Magnification of A1 and B1

L: lens, ir: iris, AC: anterior chamber

s: stroma, en: endothelial layer

7 shows the results of histological observation of the eyes of 7-month-old transgenic mice with corneal haze.

A, B: wild type mice, C, D: transgenic mice

A1 to A3, C1 to C3: hematocillin-eosin staining

B1 to B3, D1 to D3: trichrome staining

ep: epithelial layer, s: stroma

ir: iris, L: lens

8 shows the results of confirming the hβig-h3 protein and the endogenous mouse βig-h3 (mβig-h3) protein expressed in the eyes of transgenic mice with corneal haze by immunohistochemical staining.

A, C: wild type mice, B, D: transgenic mice

ep: epithelial layer, s: stroma

en: endothelial layer

The present invention relates to a transgenic mouse overexpressing βig-h3 and a method for producing the same. More specifically, the present invention relates to a transgenic mouse and a method for producing the ocular disease caused by overexpressing βig-h3 specifically in the liver and hypersecreting it into the blood.

βig-h3 (keratoepithelin) is an extracellular matrix protein induced by transforming growth factor-β (TGF-β) and first isolated from human adenocarcinoma cells (Skonier J. et al., DNA Cell Biol ., 11, 511-522, 1992), and several cell lines such as human epithelial cells, keratinocytes and fibroblasts are known to be strongly induced by TGF-β (LeBaron RG et al., J. Invest . Dermatol, 104, 844-849, 1995 ;.. Skonier J. et al, DNA Cell Biol ., 13, 571-584, 1994). In addition, βig-h3 is also found in normal human tissues such as heart, kidney and skin, and it is estimated that βig-h3 has important functions in vivo. Furthermore, βig-h3 has been reported to be strongly expressed not only in the normal cornea of human eyes but also in the healing areas of wounded corneas, and expression of corneal epithelium and the periplasm during the development of rats has been reported (Escribano). J. et al, J. Cell Physiol, 160, 511-521, 1994;..... Hirano K. et al, Curr Eye Res ., 15, 965-972, 1996; Rawe IM et al., Invest . Ophthalmol . Vis . Sci ., 38, 893-900, 1997; Ferguson JW et al., Mech . Dev . 120, 851-864, 2003).

Mutation of βig-h3 results in a genetic eye disease known as corneal dystrophies (Munier FL et al., Nat . Genet ., 15, 247-251, 1997). The corneal dystrophy is a disease in which foreign matter is deposited on the cornea, and as a result, corneal transparency is reduced to cause severe visual impairment.

On the other hand, TGF-β, which is known to induce βig-h3, is expressed mainly in the anterior part of the human eye including the cornea, thereby controlling various cell pathophysiological responses of the anterior part by controlling cell proliferation, differentiation and cell matrix composition it is known that to control (Pasquale, LR et al, Invest Ophthalmol Vis Sci, 34, 23-30, 1993;........ Song QH et al, J. Cell Biochem 77, 186-199, 2000) .

From these expressions of TGF-β and βig-h3 and their interrelationships, it is estimated that not only TGF-β but also βig-h3 play an important role in ocular disease and eye development. Therefore, the function of βig-h3 in the eye needs to be identified.

In order to elucidate the function of βig-h3 in the eye, it is very important to find a way to overexpress βig-h3 in vivo. To date, many researchers have attempted to apply plasmid DNA or adenovirus vectors directly to the eye or to administer eye drops containing them to identify the function of the particular gene in the eye. However, since these methods induce an immune response (inflammation), it has been difficult to study the function of certain genes in the eye (Tsubota K. et al., Exp . Eye Res ., 67, 531-538, 1998; Noisakran S. et al., J. Immunol ., 162, 4184-4190, 1999). To overcome this problem, Sakamoto et al. Attempted to study the function of specific proteins in the eye by injecting adenovirus vectors into the skeletal muscles of mice so that specific secreted proteins produced in muscle accumulate in the cornea along blood vessels. (Sakamoto T. et al., Gene Ther ., 7, 1915-1924, 2000).

In order to investigate the function of βig-h3 in the eye, the present inventors studied a method for overexpressing βig-h3 in vivo. The present invention was completed by obtaining a transgenic mouse overexpressing βig-h3 from the fertilized egg of the mouse and confirming that the transgenic mouse caused eye disease due to overexpression of βig-h3.

It is therefore an object of the present invention to provide a fertilized egg of a mouse in which a gene construct comprising a βig-h3 gene engineered to be specifically overexpressed in the liver, a transgenic mouse obtained from the fertilized egg, and a method of manufacturing the same.

In order to achieve the above object, the present invention provides a fertilized egg of a mouse introduced with a gene construct comprising a βig-h3 gene prepared to be specifically overexpressed in the liver.

The present invention also provides a transgenic mouse obtained by implanting the fertilized egg into the uterus of the surrogate mother.

The present invention also provides a method for producing the mouse.

Hereinafter, the present invention will be described in more detail.

The present invention provides a fertilized egg of a mouse introduced with a gene construct comprising a βig-h3 gene engineered to specifically overexpress in the liver.

The gene construct is characterized in that the liver-specific enhancer / promoter and βig-h3 gene is operably linked.

As used herein, the term “operably linked” means that the βig-h3 gene is linked in such a way as to enable expression of the gene when it is bound to an expression control sequence (enhancer / promoter). it means.

The liver specific enhancer / promoter may be used without limitation as long as it can regulate βig-h3 gene to be specifically expressed in the liver. For example, the Apolipoprotein E (ApoE) promoter, the transthyretin (TTR) promoter, the liver-specific human serum amyloid P component promoter, the albumin promoter, and the like. Preferably, albumin promoter and enhancer can be used.

The βig-h3 gene is preferably mouse βig-h3 (hereinafter referred to as mβig-h3, SEQ ID NO: 1) or human βig-h3 gene (hereinafter referred to as hβig-h3), which is very similar to the mouse βig-h3. Number 2) may be used. Such gene sequences are known in the art and are as shown in SEQ ID NO: 1 and SEQ ID NO: 2.

In addition, the gene construct may be sequentially connected to an internal ribosomal entry site (IRES), a reporter gene, an intron, and a poly A sequence downstream of the βig-h3 gene (3 ′).

The IRES may be one known in the art as a site where the ribosome binds to protein synthesis. For example, the IRES includes encephalomyocarditis virus IRES and adenovirus IRES known in the art.

The reporter gene is used to easily observe the specific expression in the liver of the βig-h3 gene. More specifically, since the reporter gene is regulated by a liver specific expression promoter, it may be used instead of directly detecting the expression of the βig-h3 gene whose expression is regulated by the same promoter, and may be used directly or indirectly. Standard methods can easily detect their activity or degree of production. For example, the reporter genes include LacZ, GFP or EGFP, RFP, hpAP (human placental alkaline phosphatase).

The intron is not particularly limited and may be a kind known in the art. For example, the mouse protamine-1 gene intron, the chicken β-globin intron, and the like. In the present invention, mouse protamine-1 gene intron was used.

The poly A sequence is not particularly limited and may be a kind known in the art. For example, from the SV40 poly A sequence, fetal growth hormone poly A sequence, mouse protamine-1 poly A signal, large T antigen polyA region Poly A signals, poly A signals derived from rabbit β-globin, and the like. In the present invention, mouse protamine-1 gene poly A sequence was used.

In an embodiment of the present invention, a gene construct in which albumin enhancer / promoter, hβig-h3 gene, and IRES-LacZ-mp1 / pA are sequentially connected was prepared and used (see FIG. 1).

Introduction of the gene construct into mouse fertilized eggs may use methods known in the art. As a method of introducing a gene into a fertilized egg, for example, (1) a microinjection technique of injecting a gene as a micromanipulation technique into a male pronucleus of a conjugate in the pronuclear stage immediately after fertilization, and (2) an embryonic stem inserting the gene into cells and implanting it into the embryo of the blastocyst (Stem cell insertion technique), (3) introducing the gene into the embryo using a retroviral vector (Retroviral insertion technique), ( 4) Injecting genes into male testes to insert genes into sperm and modify them with eggs (sperm-mediated gene transfer technique). Of these, the micro-injection method is most used.

The present invention also provides a transgenic mouse obtained by implanting a mouse fertilized egg into which the βig-h3 gene is introduced into a surrogate mother and a progeny produced from the mouse and overexpressing βig-h3.

The mouse is characterized in that βig-h3 is specifically overexpressed in the liver and the overexpressed protein is oversecreted into the blood and accumulated in the cornea causing eye disease.

In one embodiment of the present invention, the expression level of βig-h3 in liver tissue and plasma of the transgenic mice of the present invention was analyzed using Western blot and enzyme-linked immunosorbent assay (Examples <3-1> and <3). -2>). As a result, it was confirmed that βig-h3 was specifically overexpressed in the liver of the transgenic mouse of the present invention (see FIG. 3), and the concentration of βig-h3 in plasma of the transgenic mouse was 1.5-2 times higher than that of the wild type mouse. It was confirmed that the degree increased (see FIG. 4).

Meanwhile, in another embodiment of the present invention, the phenotype of the transgenic mice overexpressed βig-h3 was examined. As a result of visual observation, it was confirmed that corneal haze occurred (see FIG. 5), and histological observations showed abnormalities of the cornea, anterior eye, and the lens of the eye (see FIGS. 6 and 7). Furthermore, it was confirmed that βig-h3 protein was strongly expressed in corneal epithelial cells of transgenic mice with corneal haze through immunohistochemical staining (see FIG. 8).

From the experimental results, it could be seen that βig-h3 was overexpressed in the liver and secreted into the blood in a transgenic mouse, followed by a normal vascular system, and accumulated in the cornea through the blood vessels around the cornea. As a result, it was found that the transgenic mice overexpressing βig-h3 develop corneal dystrophy, corneal haze and anterior eye hypoplasia.

Therefore, the transgenic mouse of the present invention can be usefully used as a therapeutic research model for eye diseases such as corneal dystrophy, corneal haze and anterior eye hypoplasia.

The corneal dystrophy generally refers to a disease in which pathological deposits are abnormally accumulated in the cornea of both eyes.

The corneal haze refers to a disease in which the transparency of the cornea is reduced, causing severe visual impairment.

On the other hand, the anterior part of the vertebrate eye is formed by the generation of differentiated cells in the ectoderm and has a vitreous structure including the cornea, the iris, the ciliary body, and the lens (Pei, YE and Rhodin JA, Anat . Rec . , 168, 105-125, 1970). Anterior ocular dysplasia is a disease in which the formation of the anterior ocular region is poor.It is autosomal dominant iridogoniodysgenesis dysplasia, glaucoma with anterior chamber development disorder, congenital hereditary corneal endothelial dystrophy, iris deficiency, and Peter's malformation. anomaly) (Williams DL Eye , 7, 607-616, 1993).

The present invention also provides a method for producing a transgenic mouse overexpressing the βig-h3. The production method of the present invention comprises the steps of (a) preparing a gene construct operably linked to the liver-specific enhancer / promoter and βig-h3 gene;

(b) injecting the gene construct of (a) into a fertilized egg of a mouse;

(c) implanting the fertilized egg of (b) into the uterus of the surrogate mother to obtain a transgenic mouse.

Gene construct of step (a) is connected to the internal ribosomal entry site (IRS), reporter gene, intron and poly A sequence sequentially downstream (3 ') of βig-h3 gene, the following general cloning technology It can manufacture by. First, cDNA or genomic DNA of βig-h3 of human or mouse is prepared by reverse transcription using mRNA extract extracted from tissue expressing βig-h3, or obtained by cloning from cDNA library of human or mouse, and then IRES-LacZ- Cloning into a known vector containing mpl intron / poly A and inserting a liver specific enhancer / promoter back into the vector. In one embodiment of the present invention, an albumin enhancer / promoter was used as a liver specific enhancer / promoter (see Example 1).

In order to inject the thus prepared vector into the mouse fertilized egg, a vector must be removed using restriction enzymes, and only the genes necessary for pure expression must be isolated and purified. In one embodiment of the present invention, the expression vector prepared in the present invention was cut with restriction enzymes NotI and NruI to obtain a purified transplant gene of 8.8 kb in size (see Example 1).

Injecting the gene construct into the mouse fertilized egg in the step (b) can be carried out according to a conventional method. The preferred method is to directly inject the gene construct into the fertilized nucleus of the fertilized egg (Hogan et al., Manipulation g the mouse embryo , Cold Spring Harbor Laboratory, Cold Spring Harbor, 1994). Preferably, the fertilized egg of the mouse is a conjugate in the pronuclear stage immediately after fertilization, and the gene construct is preferably inserted into the pronucleous of the conjugate.

The injection method may use a method known in the art, and specific examples thereof are as described above.

In step (c), the fertilized egg implanted with the gene construct is implanted into the womb of a pseudo-pregnant surrogate mother. The fertility surrogate mother refers to a female mouse that is crossed with a male mouse whose resected tube was excised.

Transgenic mice obtained through the method can be determined genotype by performing DNA analysis according to methods known in the art. For example, by performing Southern blot or PCR using the tail genomic DNA of the transgenic mice obtained above, it is possible to confirm whether or not the βig-h3 gene has been introduced.

Furthermore, the genotype-transformed mice can be determined by visual observation or histological observation to determine the phenotype.

Below. The present invention will be described in detail by way of examples.

However, the following examples are merely to illustrate the invention, but the content of the present invention is not limited to the following examples.

< Example  1>

Albumin -hβ igh3  gene Constructed  Construction and Production of Transgenic Mice

To prepare transgenic mice that overexpress βig-h3, a gene for transplantation was first constructed.

Total DNA ( SEQ ID NO: 2 ) of human βig-h3 (hβig-h3) with very high identity to mouse βig-h3 was obtained from pBluescript cloned with full-length βig-h3 cDNA (Kim J.-E et al. al, J. Cell Biochem, 77: ... 169-178, 2000; Kim, J.-E. et al ., J. Biol . Chem ., 275: 30907-30915, 2000). The βig-h3 SalI / XhoI fragments (amino acids 1-683, βig-h3 overall) were cloned into the SalI site of pCD3, which was cloned with a 4.3 kb IRES-LacZ-mp1 intron / polyA. The LacZ gene was used as a reporter for verifying whether βig-h3 expression was properly performed in the liver of a mouse into which a transplanted gene was inserted. In addition, a 2.3 kb sized albumin (Albumin, Alb) enhancer / promoter fragment was cloned that can specifically express the liver at the NotI-SalI position of the vector. The Alb enhancer / promoter is known as Al. It was provided by Dr. Palmer (Dr. R. Palmiter, University of Washington, Seattle, WA). The vector was cut with NotI and NruI enzymes to obtain a purified transplant gene of 8.8 kb size, which was named Albumin-hβigh3 (FIG. 1).

The transplanted gene was microinjected into the pronucleous of a C57BL / 6 mouse fertilized egg by request from Macrogen Company (Seoul, Korea). The fertilized egg inserted with the transplanted gene was implanted in the uterus of the surrogate mother mouse to obtain 200 or more mice, and 7 original mice were obtained through PCR genotyping.

The inventors named the albumin-hβigh3 fertilized eggs of the seven original mice (transgenic mice) obtained by microinjecting an albumin-hβigh3 construct into the pronucleous of the C57BL / 6 mouse fertilized egg, and the Budapest treaty It was deposited on January 16, 2007 by the Korean Collection for Type Cultures, an international depository institution, and was awarded the accession number KCTC 11063 BP.

< Example  2>

Albumin -hβ igh3  Genetic Analysis of Transgenic Mice

Genomic DNA was extracted from the tail of a mouse obtained by crossing the original mouse obtained in Example 1 with a wild-type mouse, and PCR gene analysis was performed using a primer capable of specifically amplifying LacZ cDNA or hβig-h3 cDNA. It was.

Primers capable of specifically amplifying LacZ cDNA or hβig-h3 cDNA (SEQ ID NOS: 3 to SEQ ID NO: 6) are shown in Table 1 below, and PCR analysis showed that 100 ng, 0.2 genomic DNA was obtained from the tail of the mouse. 1 μM of primer, 1 mM dNTP mixture, 3 μl of Taq buffer and 1 Unit of Taq polymerase were mixed and performed using GeneAmp PCR System 9600 (PE Applied Biosystems, USA). PCR reaction conditions are as follows. 10 minutes denaturation at 95 ° C, 95 ° C for 1 minute, 58 ° C for 1 minute (when using LacZ cDNA primer) or 48 ° C for 1 minute (when using hβig-h3 cDNA primer), 72 ° C for 1 minute DNA amplification and finally DNA extension at 72 ° C. for 10 minutes.

Primer Sequences of LacZ cDNA and hβig-h3 cDNA primer order SEQ ID NO: LacZ Forward Primer 5'-TAATCACGACGCGCTGTATC-3 ' 3 LacZ reverse primer 5'-CGGATAAACGGAACTGGAAA-3 ' 4 forward primer of hβig-h3 5'-TCATCGATAAGGTCATCTCC-3 ' 5 reverse primer of hβig-h3 5'-CGGTTCAAAGTCTCACTAGG-3 ' 6

Complete DNA amplification was performed by 1% agarose electrophoresis. As a result, the result of 500bp when using the LacZ cDNA primer, and 202bp when using the hβig-h3 cDNA primer was confirmed (FIG. 2).

< Example  3>

Albumin -hβ igh3  Transformation In mice  hβ ig - h3 Expression verification

In the liver tissue and plasma of Albumin-hβigh3 transgenic mice, it was confirmed by Western blot and enzyme-linked immunosorbent assay.

<3-1> Weston  Blot western blot )

Liver tissues of two-month-old Albumin-hβigh3 transgenic mice and wild-type mice were prepared using RIPA buffer (150 mM NaCl, 10 mM Tris pH 7.2, 0.1% SDS, 1% Triton X-100, 1% deoxycholate, 5 mM EDTA). After dissolution, Western blots were performed using this as a sample. In the experiment, a monoclonal mouse anti-hβigh3 antibody and horseradish peroxide-conjugated goat anti-mouse IgG (horseradish peroxide-conjugated goat anti-mouse IgG) coupled with mustard peroxidase (Santa Cruz Biotechnology Biotech., Inc. USA) were used. It was. The anti-hβigh3 antibodies were prepared according to previously known methods (Kim J.-E et al., J. Biol . Chem . 275, 30907-30915, 2000).

As a result, it was confirmed that hβig-h3 protein was strongly expressed in liver tissue of Albumin-hβigh3 transgenic mice. In contrast, hβig-h3 was weakly expressed in wild-type mice (FIG. 3). From the experimental results, it was found that hβig-h3 was overexpressed in the liver of a transgenic mouse prepared according to the method of the present invention. On the other hand, the weak expression of hβig-h3 in wild-type mice was similar to that of human βig-h3 and mouse βig-h3 (mβig-h3). This is because it was detected to some extent.

<3-2> enzyme-linked immunosorbent assay ( ELISA )

In order to measure the concentration of hβig-h3 contained in the plasma of Albumin-hβigh3 transgenic mice, an enzyme-linked immunosorbent assay (ELISA, Regen Biotech, Seoul, Korea) was performed.

First use a 96-well plastic flat microtiter plate (Corning NY, USA) at 4 ° C using a coating buffer (20 mM carbonate-bicarbonate buffer, pH 9.6 with 0.02% sodium azide). Coated recombinant hβig-h3 protein overnight. Recombinant hβig-h3 protein was prepared according to previously known methods (Kim J.-E et al., J. Biol . Chem . 275, 30907-30915, 2000).

Blood was collected using potassium-EDTA-coated microbet tubes (Sarstedt, Germany) from the lower extremity saphenous veins of two-month-old wild type mice and transgenic mice, and then plasma was obtained by centrifugation at 3000 rpm at 4 ° C. for 30 minutes. . The plasma was diluted with PBS-T (saline buffered with phosphate with 0.05% Tween 20) and mixed with anti hβig-h3 antibody in 96-well round microtiter plates and reacted at 37 ° C. for 90 minutes. 15 minutes before the reaction, the plate plate coated with recombinant hβig-h3 protein was taken out at 4 ° C and washed with PBS-T three times for 5 minutes at room temperature. Plasma and antibodies reacted in the round plate above were transferred to a plate plate washed with PBS-T and reacted at room temperature for 30 minutes. After the reaction was completed, the plate was washed in the same manner and reacted at room temperature for 90 minutes by adding a peroxidase-coupled anti-rabbit IgG antibody (Santa Cruz Biotechnology, California, USA). After the reaction, the plate was washed in the same manner, and then treated with 200 μl of substrate solution (100 μg / ml ο-phenylenediamine and 0.003% H ₂ O ₂ ), followed by reaction for 60 minutes in a place where light at room temperature was blocked. 50 μl of 8N H ₂ SO ₄ was added to stop the reaction, and the absorbance was measured at 490 nm using a Bio-Rad model 550 microplate reader.

As a result, the mean value of hβig-h3 protein concentration in plasma of wild-type mice was 150ng / ml (n = 10, SD = 55). On the other hand, in the case of Albumin-hβigh3 transgenic mice, it was 292 ng / ml (n = 28, SD = 90), which was about 1.5-2 times higher than that of wild-type mice (FIG. 4). From the above results, it was confirmed that hβig-h3 was overexpressed in the blood of the transgenic mouse of the present invention.

< Example  4>

Albumin -hβ igh3  Phenotypic Survey of Transgenic Mice

Phenotypic changes in Albumin-hβigh3 transgenic mice were visually and histologically observed.

<4-1> Visual observation

Two-month-old wild-type mice and two-month-old, 12-month-old and 14-month-old transgenic mice were observed.

As a result, there was no significant difference in the size or growth of the mouse and no significant difference in the size of the eyeball. However, in the transgenic mice, corneal haze was observed in only one eye or in both eyes, and sometimes transgenic mice with poorly developed eyes were also observed (FIG. 5).

<4-2> Histological observation

To examine the corneal haze of the transgenic mice observed with the naked eye, the eyes of 2 and 7 month old transgenic mice or wild-type mice were fixed in 4% paraformaldehyde (PFA) solution for 16 hours at 4 ° C. Paraffin block was prepared by dehydration by increasing the alcohol concentration. The prepared paraffin block was sliced to a thickness of 4 μm, hematoxylin-eosin (H & E) staining and Masson's trichrome staining were performed, and then corneas were observed.

As a result of the experiment, the corneal, anterior eye and lens were found in the corneal haze of the transgenic mice compared with the wild type mice. More specifically, the cornea of two-month-old transgenic mice showed bulging corneal stroma, resulting in non-uniform collagen layer and discontinuous formation of corneal endothelium behind the cornea. It was observed. In addition, the anterior chamber of the two-month-old transgenic mouse eye was narrowed as compared with the wild-type mouse, and the epithelial cells of the lens were grown in multiple layers (FIG. 6).

Similar but more severe ocular malformations have also been identified in 7-month-old transgenic mice. That is, in the case of the 7-month-old transgenic mice, no separation between the lens and the cornea occurred. In addition, the front lens and iris were completely attached to the posterior corneal wall, and the anterior chamber was severely narrowed. The epithelial cells of the lens lens and the fibrous thin layer remained largely growing at the attachment point between the lens and the cornea (FIG. 7). The ocular posterior wall, including the sclera, showed no significant change in transgenic mice compared with wild-type mice (results not shown).

<4-3> immunohistochemical staining ( Immunohistochemistry )

Method commonly used using paraffin slides of 2 month old mice used in histological observation of Example <4-2> (Ha, SW et al., J. Cell . Biochem ., 88, 774-782, 2003 ), Immunohistochemical staining of the eyes showing obvious corneal abnormalities was performed. Paraffin-embedded ocular tissues were removed from the slides cut to 4 μm thickness, and then treated with a 10% H ₂ O ₂ solution diluted with PBS to remove the peroxidase activity present in the tissues. Tissue slides were placed in 1 mM Tris solution (pH 9.0) containing 0.5 mM EGTA and heated in the microwave for 10 minutes to expose the antibodies. After blocking with PBS containing 1% BSA, 0.05% saponin, 0.2% gelatin, tissue slides were reacted overnight at 4 ° C. with polyclonal rabbit anti-hβig-h3 antiserum or polyclonal rabbit anti-mouse βig-h3 antiserum. I was. The tissue slides were washed three times and then reacted with horseradish peroxide-conjugated goat anti-rabbit IgG (DAKO, Glostrup, Denmark) for 90 minutes at room temperature. Coloring with diaminobenzidine tetrahydrochloride (DAB, DAKO) solution and counterstaining with hematoxylin (Mayer, Sigma). Normal rabbit IgG was used as a negative control.

As a result, it was observed that hβig-h3 protein was strongly expressed in corneal epithelial cells of transgenic mice with corneal haze (FIG. 8B). In contrast, hβig-h3 protein was not expressed at all in the normal corneal stroma squamous cells of wild-type mice (FIG. 8A). This suggests that hβig-h3 protein, which is overexpressed in the liver and secreted into the blood, has accumulated along the normal vascular system and accumulated in the cornea through the blood vessels around the cornea. Meanwhile, it was observed that mβig-h3 protein was expressed in the corneal innate of wild type mice and transgenic mice with corneal haze (FIG. 8C and D).

As described above, the transgenic mice of the present invention can be usefully used for the study of eye development and can be very useful as a therapeutic model for eye diseases such as corneal dystrophy, corneal haze and anterior eye hypoplasia.

<110> Kyungpook National University Industry-Academic Cooperation Foundation <120> Transgenic mice overexpressing betaig-h3 and method for producing          about <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 2674 <212> DNA <213> Mus musculus <400> 1 ggcacgagcc tgctttcatc gtgggtccgc gcgtgctcca gctccatggc gctcctcatg 60 cgactgctga ccctcgctct ggcactgtct gtgggccccg ctgggaccct tgcaggtccc 120 gccaagtcac cctaccagct ggtgctgcag catagccggc tccggggtcg ccagcacggc 180 cccaatgtat gtgctgtgca gaaggtcatt ggcaccaaca agaaatactt caccaactgc 240 aagcagtggt accagaggaa gatctgcggc aagtcgacag tcatcagtta tgagtgctgt 300 cctggatatg aaaaggtccc aggagagaaa ggttgcccag cagctcttcc gctctcaaat 360 ctgtatgaga ccatgggagt tgtgggatcg accaccacac agctgtatac agaccgcaca 420 gaaaagctga ggcctgagat ggagggaccc ggaagcttca ccatctttgc tcctagcaat 480 gaggcctggt cttccttgcc tgcggaagtg ctggactccc tggtgagcaa cgtcaacatc 540 gaactgctca atgctctccg ctaccacatg gtggacaggc gggtcctgac cgatgagctc 600 aagcacggca tgaccctcac ctccatgtac cagaattcca acatccagat ccatcactat 660 cccaatggga ttgtaactgt taactgtgcc cggctgctga aggctgacca ccatgcgacc 720 aacggcgtgg tgcatctcat tgacaaggtc atttccacca tcaccaacaa catccagcag 780 atcattgaaa tcgaggacac ctttgagaca cttcgggccg ccgtggctgc atcaggactc 840 aataccgtgc tggagggcga cggccagttc acactcttgg ccccaaccaa cgaggccttt 900 gagaagatcc ctgccgagac cttgaaccgc atcctgggtg acccagaggc actgagagac 960 ctgctaaaca accacatcct gaagtcagcc atgtgtgctg aggccattgt agctggaatg 1020 tccatggaga ccctgggggg caccacactg gaggtgggct gcagtgggga caagctcacc 1080 atcaacggga aggctgtcat ctccaacaaa gacatcctgg ccaccaacgg tgtcattcat 1140 ttcattgatg agctgcttat cccagattca gccaagacac tgcttgagct ggctggggaa 1200 tctgacgtct ccactgccat tgacatcctc aaacaagctg gcctcgatac tcatctctct 1260 gggaaagaac agttgacctt cctggccccc ctgaattctg tgttcaaaga tggtgtccct 1320 cgcatcgacg cccagatgaa gactttgctt ctgaaccaca tggtcaaaga acagttggcc 1380 tccaagtatc tgtactctgg acagacactg gacacgctgg gtggcaaaaa gctgcgagtc 1440 tttgtttatc gaaatagcct ctgcattgaa aacagctgca ttgctgccca tgataagagg 1500 ggacggtttg ggaccctgtt caccatggac cggatgttga cacccccaat ggggacagtt 1560 atggatgtcc tgaagggaga caatcgtttt agcatgctgg tggccgccat ccagtctgca 1620 ggactcatgg agatcctcaa ccgggaaggg gtctacactg tttttgctcc caccaatgaa 1680 gcgttccaag ccatgcctcc agaagaactg aacaaactct tggcaaatgc caaggaactt 1740 accaacatcc tgaagtacca cattggtgat gaaatcctgg ttagcggagg catcggggcc 1800 ctggtgcggc tgaagtctct ccaaggggac aaactggaag tcagctcgaa aaacaatgta 1860 gtgagtgtca ataaggagcc tgttgccgaa accgacatca tggccacaaa cggtgtggtc 1920 tatgccatca acactgttct gcagccgcca gccaaccgac cacaagaacg aggagatgag 1980 ctggcagact ctgcccttga aatcttcaaa caggcgtcag cgtattccag ggctgcccag 2040 aggtctgtgc gacttgcccc tgtctatcag cggttactgg agaggatgaa gcattagcag 2100 gaagaccgag gaggagagcc ctgcagcagc ttcccgccag tttctctcag tttgccaaag 2160 agaccattga atgtttttga aaccaaagag cacacttcaa catacatggg cgcaccatat 2220 tgagatctga gccttggacg ggtagggaag gggttaaggg gagaaaggtt ctttttagct 2280 ttgatccctc caaaccgtgg ttgttaaccc attcgaatat acagatctgg cagtcatagc 2340 ttggcaccaa attcccgaaa gacctctcga aagcatgaat ttcctgactg tgccaaggcc 2400 tgataaaggg aactacggca tcttggagct cacaaatgtg aatcaagcag tccggcattc 2460 tggaaagcct tggcatggtt ctgtaaagct cttgtaccgc tggagaaacg gcatcactat 2520 aagctatgag ttgaactgtt tctgtcaagt atgtcttgtg tccacacatg gtttggatgc 2580 ttctatattg gccctgccca ggtagaaagg gtaagaagaa catgtagaat ccagattccc 2640 tgagtgtgag ggacccatgg tgcatttgta ataa 2674 <210> 2 <211> 2691 <212> DNA <213> Homo sapiens <400> 2 gcttgcccgt cggtcgctag ctcgctcggt gcgcgtcgtc ccgctccatg gcgctcttcg 60 tgcggctgct ggctctcgcc ctggctctgg ccctgggccc cgccgcgacc ctggcgggtc 120 ccgccaagtc gccctaccag ctggtgctgc agcacagcag gctccggggc cgccagcacg 180 gccccaacgt gtgtgctgtg cagaaggtta ttggcactaa taggaagtac ttcaccaact 240 gcaagcagtg gtaccaaagg aaaatctgtg gcaaatcaac agtcatcagc tacgagtgct 300 gtcctggata tgaaaaggtc cctggggaga agggctgtcc agcagcccta ccactctcaa 360 acctttacga gaccctggga gtcgttggat ccaccaccac tcagctgtac acggaccgca 420 cggagaagct gaggcctgag atggaggggc ccggcagctt caccatcttc gcccctagca 480 acgaggcctg ggcctccttg ccagctgaag tgctggactc cctggtcagc aatgtcaaca 540 ttgagctgct caatgccctc cgctaccata tggtgggcag gcgagtcctg actgatgagc 600 tgaaacacgg catgaccctc acctctatgt accagaattc caacatccag atccaccact 660 atcctaatgg gattgtaact gtgaactgtg cccggctcct gaaagccgac caccatgcaa 720 ccaacggggt ggtgcacctc atcgataagg tcatctccac catcaccaac aacatccagc 780 agatcattga gatcgaggac acctttgaga cccttcgggc tgctgtggct gcatcagggc 840 tcaacacgat gcttgaaggt aacggccagt acacgctttt ggccccgacc aatgaggcct 900 tcgagaagat ccctagtgag actttgaacc gtatcctggg cgacccagaa gccctgagag 960 acctgctgaa caaccacatc ttgaagtcag ctatgtgtgc tgaagccatc gttgcggggc 1020 tgtctgtaga gaccctggag ggcacgacac tggaggtggg ctgcagcggg gacatgctca 1080 ctatcaacgg gaaggcgatc atctccaata aagacatcct agccaccaac ggggtgatcc 1140 actacattga tgagctactc atcccagact cagccaagac actatttgaa ttggctgcag 1200 agtctgatgt gtccacagcc attgaccttt tcagacaagc cggcctcggc aatcatctct 1260 ctggaagtga gcggttgacc ctcctggctc ccctgaattc tgtattcaaa gatggaaccc 1320 ctccaattga tgcccataca aggaatttgc ttcggaacca cataattaaa gaccagctgg 1380 cctctaagta tctgtaccat ggacagaccc tggaaactct gggcggcaaa aaactgagag 1440 tttttgttta tcgtaatagc ctctgcattg agaacagctg catcgcggcc cacgacaaga 1500 gggggaggta cgggaccctg ttcacgatgg accgggtgct gaccccccca atggggactg 1560 tcatggatgt cctgaaggga gacaatcgct ttagcatgct ggtagctgcc atccagtctg 1620 caggactgac ggagaccctc aaccgggaag gagtctacac agtctttgct cccacaaatg 1680 aagccttccg agccctgcca ccaagagaac ggagcagact cttgggagat gccaaggaac 1740 ttgccaacat cctgaaatac cacattggtg atgaaatcct ggttagcgga ggcatcgggg 1800 ccctggtgcg gctaaagtct ctccaaggtg acaagctgga agtcagcttg aaaaacaatg 1860 tggtgagtgt caacaaggag cctgttgccg agcctgacat catggccaca aatggcgtgg 1920 tccatgtcat caccaatgtt ctgcagcctc cagccaacag acctcaggaa agaggggatg 1980 aacttgcaga ctctgcgctt gagatcttca aacaagcatc agcgttttcc agggcttccc 2040 agaggtctgt gcgactagcc cctgtctatc aaaagttatt agagaggatg aagcattagc 2100 ttgaagcact acaggaggaa tgcaccacgg cagctctccg ccaatttctc tcagatttcc 2160 acagagactg tttgaatgtt ttcaaaacca agtatcacac tttaatgtac atgggccgca 2220 ccataatgag atgtgagcct tgtgcatgtg ggggaggagg gagagagatg tactttttaa 2280 atcatgttcc ccctaaacat ggctgttaac ccactgcatg cagaaacttg gatgtcactg 2340 cctgacattc acttccagag aggacctatc ccaaatgtgg aattgactgc ctatgccaag 2400 tccctggaaa aggagcttca gtattgtggg gctcataaaa catgaatcaa gcaatccagc 2460 ctcatgggaa gtcctggcac agtttttgta aagcccttgc acagctggag aaatggcatc 2520 attataagct atgagttgaa atgttctgtc aaatgtgtct cacatctaca cgtggcttgg 2580 aggcttttat ggggccctgt ccaggtagaa aagaaatggt atgtagagct tagatttccc 2640 tattgtgaca gagccatggt gtgtttgtaa taataaaacc aaagaaacat a 2691 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for LacZ <400> 3 taatcacgac gcgctgtatc 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for LacZ <400> 4 cggataaacg gaactggaaa 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> forward primer for h betaig-h3 <400> 5 tcatcgataa ggtcatctcc 20 <210> 6 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> reverse primer for h betaig-h3 <400> 6 cggttcaaag tctcactagg 20  

Claims (10)

Albumin enhancer / promoter, βig-h3 gene represented by the nucleotide sequence of SEQ ID NO: 2 and Internal Ribosomal Entry Site (IRS), LacZ gene, mouse protamine-1 gene intron and poly in sequence at the 3 'end of the βig-h3 gene Fertilized egg (KCTC 11063BP) of a mouse in which the gene construct described in FIG. 1 is operably linked to sequence A. delete delete delete A transgenic mouse obtained by implanting the fertilized egg of claim 1 into the uterus of a surrogate mother. The transgenic mouse of claim 5, wherein the transgenic mouse is overexpressed in βig-h3 represented by the nucleotide sequence of SEQ ID NO: 2. 7. The transgenic mouse according to claim 5, wherein the transgenic mouse has an ocular disease. 8. The transgenic mouse according to claim 7, wherein the ocular disease is selected from the group consisting of corneal dystrophy, corneal haze and anterior ocular dysplasia. Progeny produced from the mouse of claim 5, overexpressing βig-h3 represented by the nucleotide sequence of SEQ ID NO: 2. (a) a gene construct in which an albumin enhancer / promoter, a βig-h3 gene represented by the nucleotide sequence of SEQ ID NO: 2, an internal ribosomal entry site (IRS), a LacZ gene, a mouse protamine-1 gene intron, and a poly A sequence are sequentially linked Preparing a tray; (b) injecting the gene construct of (a) into a fertilized egg of a mouse to prepare a fertilized egg of a mouse having an accession number of KCTC 11063BP; And (c) a method of producing a transgenic mouse overexpressing βig-h3 represented by the nucleotide sequence of SEQ ID NO: 2 comprising the step of implanting the fertilized egg of (b) into the uterus of a surrogate mother to obtain a transgenic mouse.

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