KR20040039729A - Transgenic mice inducing hepatocellular carcinoma by the expression of mutant type H-ras cDNA - Google Patents

Abstract

PURPOSE: A transgenic mouse inducing hepatocellular carcinoma is provided, thereby overexpressing mutant type H-ras gene and inducing hepatocellular carcinoma, so that it can be useful for screening medicines for treating the liver cancer or for studying on the liver cancer incidence mechanism. CONSTITUTION: A method for producing a transgenic mouse inducing hepatocellular carcinoma comprises the steps of: constructing a H-ras expression vector containing 5' flanking sequence with a mouse albumin enhancer/promoter, human H-ras cDNA in which a 12th codon is mutated from glycine to valine, and a SV40 poly A sequence for transcription termination; micro-injecting the H-ras expression vector into a fertilized mouse egg; transplanting the mouse egg into a mouse oviduct; and growing the mouse egg, wherein the hepatocellular carcinoma is induced in a 6-month-old mouse.

Description

Transgenic mice inducing hepatocellular carcinoma by the expression of mutant type H-ras cDNA}

The present invention relates to liver cancer-expressing transgenic mice, and more particularly, by introducing the H-ras gene in which the twelfth codon is substituted with glycine to valine to produce liver cancer-expressing transgenic mice. As liver cancer is overexpressed, the present invention relates to a liver cancer-expressing transgenic mouse that can be usefully used in animal experiments when developing new drugs, and also useful for studying the pathogenesis of liver cancer and discovering new genes involved in the development of liver cancer.

H-ras (H-ras) is an oncogene present in high frequency in active forms in tissues such as liver cancer. Therefore, the expression of H-ras is thought to lead to the development of liver cancer, and attempts to develop cancer-causing transgenic mice using the H-ras gene were as follows.

Sandgren et al. [Oncogene 1989, 4, 715-724] produced transgenic mice using a vector DNA with an active H-ras gene 12 kb linked to an albumin enhancer / promoter. It had an unusual liver structure, but died a few days after birth, and another group reported hepatic dysplasia but eventually died of lung cancer.

In addition, Tremblay et al. [Mol. cell. Biol., 1989 9, 854-859, reported that the production of murine mammary tumor virus (MMTV) H-ras transgenic mice resulted in a relatively late progression of tumors in various tissues.

Meanwhile, Gilbert et al. [Int. J. Cancer 1997, 73, 749-756] described the human H-ras oncogene (12th codon) under the control of the L-type pyruvate kinase gene 5 'flanking sequence. Mutant) produced transgenic mice that overexpressed, resulting in liver cancer, including renal impairment, but males reported infertility.

In addition, Hayashi et al. [Toxicol Pathol 1998, 26 (4): 556-61] reported that 5 out of 244 transgenic mice (rasH2) overexpressing human cH-ras caused liver cancer. Only a few mice older than 10 years have liver cancer, which is very rarely used as a liver cancer mouse.

As such, transgenic mice overexpressing H-ras have been developed, but due to problems such as premature death, infertility, or low liver cancer-causing ability, they were not suitable as disease model animals for drug efficacy testing of new drug candidates. Therefore, it is necessary to invent an experimental animal that causes liver cancer at a suitable time, does not cause lesions in other organs, and reproduces well.

The reason why the transgenic mice overexpressing the above-mentioned active H-ras gene is not suitable for use as a disease model animal is that the side effects such as premature death are caused due to the overexpression of the active H-ras protein. In other words, they used the H-Ras gene of genomic DNA as a reporter gene, and in general, transgenic animals used genomic DNA rather than cDNA. It is known that the expression level is 5 to 10 times higher. Therefore, the present inventors have tried to lower the expression level by using the H-ras cDNA (12th codon is substituted with glycine in glycine) as a reporter gene of the expression vector. That is, a recombinant expression vector was prepared by inserting a fusion gene consisting of an albumin enhancer / promoter, an H-ras cDNA (the twelfth codon substituted by glycine to valine), and an SV40 poly A into a pBluescript vector, and constructing a pBluescript of the expression vector. The vector portion was removed, and only the fusion gene was purified and injected into the fertilized egg of the mouse, and implanted into the fallopian tube of the surrogate mother to continue development. As a result, the twelfth codon of the H-Ras gene lowered the expression level using glycine-substituted cDNA, preventing premature death of mice due to overexpression of active H-Ras, and low H-Ras It was confirmed that the expression level, hepatic cancer was generated well at 6 months of age, the present invention was completed by confirming the good reproduction.

Accordingly, an object of the present invention is to provide a transgenic mouse that causes H-ras liver cancer.

1 is a schematic diagram showing the manufacturing process of the expression vector A / ras produced for the production of liver cancer-expressing transgenic mice.

Figure 2 is a photograph confirmed by PCR that the vector DNA is inserted on the chromosome of the transgenic mouse.

Figure 3 is a photograph confirmed by RT-PCR transcripts expressed in each tissue of liver cancer-expressing transgenic mice.

Figure 4 is a photograph confirmed by the Northern blot (Northern blot) expressed in liver tissue of liver cancer-expressing transgenic mice.

Figure 5 is a photograph confirming the H-ras (H-ras) protein expressed in liver tissue of liver cancer-expressing transgenic mice by Western blot.

Figure 6 shows a macroscopic picture (A) and microscopic pictures (B: normal liver tissue, C: liver cancer tissue) after induction of liver cancer in liver cancer-expressing transgenic mice.

The present invention is characterized by recombinant expression vectors and H-ras cDNAs in which the 12th codon is substituted with valine in glycine and E-ras transgenic mice.

The present invention will be described in more detail as follows.

The present invention is to solve the problems such as early death, infertility, or low liver cancer-causing ability of the transgenic mice using the existing H-Ras gene, H-ras (H-Hs) in which the twelfth codon is substituted with glycine to valine -ras) By introducing an expression vector carrying the gene into mouse fertilized eggs, H-ras were expressed at low levels, preventing premature death of mice due to overexpression of active H-ras, and well-producing liver cancer at 6 months of age. The present invention relates to a liver cancer-expressing transgenic mouse suitable for developing a liver cancer therapeutic agent by confirming that breeding is well.

The present invention aims at developing a liver cancer specific disease model transgenic mouse, and has developed a transgenic mouse in which an active H-ras cDNA is overexpressed at an appropriate level by controlling the action of an albumin enhancer / promoter to induce liver cancer. The transgene required for the production of the transgenic mouse is a human H-ras cDNA (12th codon vaginated from glycine) downstream of the 5 'flanking sequence 1.1 kb containing the mouse albumin enhancer / promoter. SEQ ID NO: 1) 0.57 kb is linked, SV40 poly A 890 bp is linked for transcription termination. A fusion gene consisting of mouse albumin enhancer / promoter / H-ras cDNA / SV40 poly A was inserted into a pBluescript vector to produce a recombinant expression vector, which was named A / ras. The total length is 5.48 kb. For microinjection into mouse fertilized eggs, the pBluescript vector portion of the expression vector was removed and only the fusion gene was purified.

In order to produce a transgenic mouse, micro-injected DNA was injected into the fertilized egg of the mouse and transplanted into the fallopian tube of the surrogate mother to obtain a litter, and obtained genomic DNA to determine whether the transgenic mouse was PCR. In addition, RNA was extracted from the tissues of the F1 transgenic mice to confirm the transcriptional expression of H-Las by RT-PCR and Northern blot, and the expression of H-Las protein by Western blot. It was confirmed. The presence of liver cancer was examined by histopathological assay. The production of liver cancer was also confirmed in the offspring by the above method.

Therefore, the liver cancer-expressing transgenic mice thus produced can be very useful as a disease model animal suitable for the study of the pathogenesis of liver cancer as well as the drug efficacy test for the development of liver cancer therapeutics.

Hereinafter, the present invention will be described in more detail based on Examples, but the present invention is not limited to the following Examples.

Example 1: Preparation of H-ras expression vector

The transgene required for the production of transgenic mice is a human H-las cDNA (12th codon glycine 12 kb downstream of the 5 'flanking sequence 1.1 kb with mouse albumin enhancer / promoter). Was replaced with a valine) 0.57 kb was linked, the fusion gene was inserted into the SV40 poly A 890 bp inserted for transcription termination (see Fig. 1).

The fusion gene was inserted into a pBluescript KSII vector to produce a recombinant expression vector of 5,480 bp and named as A / ras. The expression vector was digested with BamH1 / EcoRV to isolate and purify only the fusion gene, and then used as an injection DNA for the production of transgenic mice.

Example 2: Production of A-Ras Transgenic Mice

Microinjection of the injected DNA was performed according to a conventional method. That is, 4 ng / μg of injected DNA was microinjected into fertilized eggs of BCF1 mice, and fertile eggs in good condition were implanted into the fallopian tubes to obtain 235 litters. In order to confirm that the DNA injected into the fertilized egg was inserted on the born mouse chromosome, genomic DNA was extracted from the toes of the 10-day-old mountain and subjected to PCR. The primer used at this time is as follows.

Upstream primer (SEQ ID NO: 2)

5'-CTAGGGCTGCAGGAATTC-3`

Downstream primer (SEQ ID NO: 3)

5'-GTAGTTTAACACATTATACACT-3`

PCR conditions were first determined for DNA denaturation. 10 min, second reaction (35 ° C, 94 ° C 1 minute, 56 ° C 1 minute, 72 ° C 1 minute) for DNA amplification, and 72 ° C 1 minute for extension, the amplified DNA was stored at 4 ° C. , 0.8% agarose electrophoresis was performed to determine whether the transgenic mice (see Table 1 and Figure 2). As a result, three Founder mice (# 28, # 76, # 78) were identified. # 28 (FIG. 2) and # 76 (data not shown) were able to breed offspring because of good breeding, but # 78 was no longer able to get offspring because of infertility. As mentioned in Example 4 below, # 28 reproduces well and generates liver cancer specifically at 6 months of age. Therefore, two-cell fertilized eggs were transferred to the Korea Biotechnology Research Institute Gene Bank, July 30, 2002 for phylogenesis. Was deposited [Accession Number: KCTC 10318BP].

Injected gene Donor Number of fertilized eggs injected Number of implanted eggs Sanja Number of transformed mice A-ras BCF1 1562 1114 (71.3%) 235 3 (1.3%)

Example 3: Expression Assay of H-Ras in A-Ras Transgenic Mice

Expression of Ras in the liver of A-Ras transgenic mice was confirmed by RT-PCR, Northern blot, and Western blot.

1) RT-PCR

Total RNA was extracted from each tissue of # 28 and # 76 descendant mice by a conventional method, and the degree of transcription of the injected DNA was confirmed by a conventional RT-PCR method. That is, cDNA is synthesized according to "Reverse Transcription System [Promega Corporation]", and PCR is 94 ??. After 10 minutes of denature, (94 ° C 1 minute / 60 ° C 1 minute / 72 ° C 10 minutes) DNA amplification (35 times), 72 ° C 10 minutes extension reaction and stored at 4 ° C . G3PDH was used as internal control of RT-PCR. As a result, as shown in FIG. 3, the band was confirmed in liver tissue, and # 76 (data not shown), the transcription was confirmed in most tissues tested.

2) Northern blot

RT-PCR results were confirmed by conventional Northern blot. A probe was used after labeling [??- ³² P] dCTP of H-ras cDNA amplified using the primers shown in Example 2. The membrane used was Zeta-Probe GT blotting membranes [BIO-RAD], hybridization was allowed to react overnight at 60 ° C., and membrane washing was performed at 20 mM sodium phosphate [pH]. 7.2] / 1% SDS, washed twice at 65 ° for 10 minutes. As a result, H-Ras was strongly expressed in hepatic tissue, and weak expression was also observed in other heart tissues [FIG. 4].

3) Western blot

Experiments were carried out according to conventional methods, and the antibodies used were polyclonal rabbit anti-H-ras (C-20) antibodies [Santa Cruz Biotech., Inc.]. U.S.] and horseradish peroxidase-conjugated goat anti-rabbit IgG (Santa Cruz Biotechnology Biotech., Inc.). U.S.]. Western blot showed strong expression of H-Ras protein in liver tissue of # 28 transgenic mice, and weak expression of H-Ras in control, which is a mouse endogenous Ras protein [FIG. 5]. At the same age, it was confirmed that H-ras protein expression was increased in males than in females of transgenic mice.

Example 4: Pathological Observation of A-Ras Transgenic Mice

Histopathological observations of the tissues of # 28 and # 76 mice showed hepatic cancer-related lesions in the liver tissues of the mice, and no abnormal lesions were found in other tissues. As shown in the following Table 2, liver cancer was visually observed in most mice from 6 months of age, as shown in Table 2, and dysplasia and nodule, which are symptoms of pre-stage liver cancer, were also identified. This lesion was identified in F2 as well as in F1 according to Mendel's law. 6 shows a macroscopic photograph (A) and a microscopic photograph (C) of liver cancer.

The # 76 mice were 8 months of age, and hepatocarcinoma was observed in 6-month-old # 28 mice (data not shown), indicating that the lesion progressed 2 to 3 months later.

Cycle Dysplasia (%) nodule(%) tumor(%) 3-5 months 1/3 (33.3) 0/3 0/3 6 to 8 months 5/5 (100) 5/5 (100) 5/5 (100) 9-11 months 8/8 (100) 8/8 (100) 7/8 (87.5)

As described above, the present invention relates to a transgenic mouse overexpressing H-ras, and the DNA microinjected into the fertilized egg was inserted into the chromosome of the born live, and it was confirmed that the gene was expressed. According to the expression of 6 months of age, hepatic cancer and lesions involved in the development of liver cancer were generated, and also inherited genotypes well in descendants. Therefore, the A-ras transgenic mice according to the present invention are expected to be very useful as a disease model animal suitable for the study of the pathogenesis of liver cancer as well as the drug efficacy test for the development of liver cancer therapeutics.

Claims (2)

Recombinant expression vector incorporating H-ras cDNA in which the twelfth codon is substituted with glycine in valine. A-ras transgenic mice [KCTC 10318BP], characterized in that liver cancer is generated at 6 months of age by transformation using the expression vector of claim 1.