KR20020039705A - A Transgenic Mouse with Human Papillomavirus Type 16 E6/E7 Gene and Method of Producing it - Google Patents

Abstract

PURPOSE: A man papilloma virus 16E6/E7 transgenic mouse and a manufacturing method thereof is provided to develop a new treatment medicine by finding out the cause of disease related with the man papilloma virus 16. CONSTITUTION: The man papilloma virus 16E6/E7 transgenic mouse is manufactured by implanting a recombinant manifestation vector containing a man papilloma virus 16E6E7 gene connected to a promoter for manifesting the man papilloma virus 16E6E7 gene into a reproductive cell or a somatic. More particularly, the promoter is a tyrosinase promoter, and the recombinant manifestation vector is TYR-HPV16E6E7.

Description

A transgenic mouse with human papillomavirus type 16 e6 / e7 gene and method of producing it}

The present invention relates to a human papilloma virus type 16 E6 / E7 gene transplanted mouse and a method of manufacturing the same, and more particularly, to a germ cell and a somatic cell, which is connected to a promoter for expressing the human papilloma virus type 16 E6 / E7 gene. A human papilloma virus type 16 E6 / E7 transgenic mouse carrying a recombinant expression vector comprising a human papilloma virus type 16 E6 / E7 gene and a method of manufacturing the same.

Transgenic mice micro-inject a specific gene fragment into the fertilized egg of the mouse, implant it into the uterus of another mouse and give birth to it, and then examine the tail DNA of the mouse about 3 weeks ago and insert the gene from the outside into the mouse's DNA. It is born through the process of checking whether it is inserted into the Transgenic mice are a way to recognize the function of genes in vivo, and are expected to develop models for various diseases of the human body, develop various therapeutic agents, and develop bio-reactors that make the body of the mouse a living plant. to be.

New models of transgenic mouse technology are currently being developed as an approach to genetic problems such as cancer, and the c-myc oncogene by Professor P. Leader at Harvard University The success of the development of the implanted transgenic mouse has received great attention in that it is a model of human leukemia, and has obtained the first patent in the world for living life (US Patent No. 5, 087, 571). Subsequently, mature T-cells invented by male mice (US Pat. No. 5,175,383), Paulus JA Krimpenfort, etc., in which the prostate was enlarged by Philip Leader et al. Mice (US Pat. No. 5,175,384) and mice producing human beta interferon (US Pat. No. 5,175,385) invented by Thomas E. Wagner and the like have been introduced.

Human papilloma virus is a kind of Papovaviridea, a DNA virus of about 8 Kb. About 100 species of human papilloma virus are known to mainly form benign tumors, nodules or malignant tumors when infected with humans. Among the major causes of genital cancer, cervical cancer, and skin cancer of men known as malignant tumors, 16 and 18 types of human papillomaviruses are closely related.

Cervical cancer occurs in 1 out of 4 female cancers in Korea, and it is the most common cause of cancer among women. In particular, human papilloma virus type 16 is recognized as a major risk factor for cervical cancer, and various studies are being conducted to establish diagnostic technology, develop diagnostics, preventive vaccines, and develop therapeutic vaccines. Recently, E6 and E7 proteins of type 16 virus have been reported to play a major role in deactivating cells by inactivating them by binding to intracellular proteins p53 and Rb proteins, respectively.

The present inventors were studying the effect of the expression of E6 / E7 protein of human papillomavirus type 16 on the skin, and the mouse was transfected with a recombinant expression vector of human papilloma virus type 16 E6 / E7 linked to the tyrosinase promoter. In the case of transplantation into the skin, the skin disease occurs naturally and the skin cancer incidence test was found that the incidence of skin papilloma is much higher than that of normal mice, thereby completing the present invention.

Therefore, the object of the present invention is that the skin is naturally overproliferated and the skin papilloma incidence rate in the skin cancer-induced test is very high compared to that of a normal mouse, and thus the mechanism of development of diseases related to human papillomavirus type 16 E6 / E7 gene The present invention provides a human papilloma virus type 16 E6 / E7 transgenic mouse that can be usefully used in research fields such as identification, development of therapeutic agents, and new drug assay system, and a method of manufacturing the same.

1 is a schematic depiction of a recombinant expression vector constructed to express a human papilloma virus type 16 E6 / E7 gene under the control of a tyrosinase promoter and a tyrosinase expression gene expressing groping.

Figure 2 shows the results of PCR analysis of the tail DNA of the transgenic mouse of the present invention.

Figure 3 is a photograph of the back skin tissue of the transgenic mouse of the present invention.

Figure 4 is a result of analyzing the expression of the gene transplanted for the protein extract in each tissue of the transgenic mouse of the present invention by Western blot (western blot).

Figure 5 shows the results of papilloma-induced experiments by the chemicals of the transgenic mice of the present invention.

Figure 6 is a photograph of the transgenic mouse of the present invention induced papilloma.

In order to achieve the above object, the present invention is a recombinant expression comprising a human papilloma virus type 16 E6 / E7 gene linked to a promoter for expressing human papilloma virus type 16 E6 / E7 gene in germ cells and somatic cells Human papilloma virus type 16 E6 / E7 transgenic mice with vectors are provided.

The present invention also provides a recombinant expression vector comprising a human papilloma virus type 16 E6 / E7 gene linked to a promoter for expressing human papilloma virus type 16 E6 / E7 gene, and introducing the recombinant expression vector into a mouse fertilized egg. In addition, the present invention provides a method for producing human papillomavirus type 16 E6 / E7 transgenic mice comprising culturing a double healthy fertilized egg and inserting it into the oviduct of a surrogate mother mouse.

Hereinafter, the present invention will be described in detail.

The present invention relates to human papilloma virus type 16 having a recombinant expression vector comprising human papilloma virus type 16 E6 / E7 gene linked to a promoter for expressing human papilloma virus type 16 E6 / E7 gene in germ cells and somatic cells. A recombinant expression vector comprising a human papilloma virus type 16 E6 / E7 gene linked to an E6 / E7 gene-transplanted mouse and a promoter for expressing human papilloma virus type 16 E6 / E7 gene was prepared, and the recombinant expression vector was prepared. It consists of introducing into a fertilized egg of a mouse, culturing a healthy fertilized egg and inserting it into the oviduct of the mouse to produce a transgenic mouse carrying human papilloma virus type 16 E6 / E7 genes in germ cells and somatic cells.

In the above cases, the tyrosinase promoter is preferable as a promoter of the expression vector for expressing the human papilloma virus type 16 E6 / E7 gene.

In addition, in the above cases, the insertion efficiency of the foreign gene is excellent in linear DNA form, so that the recombinant expression vector is human papilloma virus type 16 E6 / E7 gene and SV40 poly linked to the tyrosinase promoter. It is preferable that the linear gene fragment (FIG. 1) consisting of the poly A tail gene.

Furthermore, in the above cases, a tyrosinase minigene (FIG. 1), which is a tyrosinase expression gene, together with the recombinant expression vector can be simultaneously inserted into a fertilized egg of a mouse so as to easily identify whether the gene is transplanted with the naked eye. It is desirable to insert and produce transgenic mice with black hair color.

Through the above technical configuration, the human papillomavirus type 16 E6 / E7 transgenic mice line which is naturally overproliferated in the skin was obtained, and in the case of tyrosinase minigenes being transplanted together Since the transgenic mouse strain has a dark hair color, it was easy to check whether the gene was transplanted with the naked eye, and skin papilloma (FIG. 6) occurred in more than 65% of mice at 15 weeks when the mouse was treated with chemicals. This may be useful in the field of research, such as the identification of the mechanism of development of diseases associated with human papilloma virus type 16, the development of therapeutic agents and the new drug assay system.

The following examples illustrate the present invention in detail, but do not limit the scope of the present invention.

Example 1 Preparation of Mice Implanted with Human Papilloma Virus Type 16 E6 / E7 Gene

Step 1: Construction of Human Papilloma Virus Type 16 E6 / E7 Gene Expression Vector

In order to obtain human papilloma virus (HPV) type 16 E6 / E7, the E6 and E7 sites of MMTV-HPV16 E6-E7 (Gen Kondoh et al., 1991, J. Virol., 65, 3335-3339) were PCR amplified and 800bp. DNA fragments were obtained. PCR analysis and primers used were performed in the same manner as PCR analysis during transgenic mice.

A 2.47 Kb tyrosinase promoter obtained by treatment of Tyrosinase minizine (Takahiko Yokoyama et al., 1990, Nucleic Acids Res., 18, 7293-7298) with EcoRI and SalI was obtained. The two fragments were ligated to obtain 3.27 Kb fragments, which were then cloned into the EcoRI and SmaI restriction sites of the pSI vector purchased from Promega to prepare pTyr-HPV16E6E7 and transformed into DH5α E.coli. Stored. To prepare the microinjected DNA solution, the stored E. coli was inoculated in LB medium (1% Bactotryton, 0.5% Yeast Extract, 1% NaCl) containing ampicilline, and then incubated overnight at 37 ° C. , The grown bacteria were collected by centrifugation at 2,000g for 10 minutes, and dissolved in 50mM glucose, 10mM Tris-Cl, 1mM EDTA, 4mg / ml lysozyme solution, and left for 5 minutes on ice. Again, add 2 volumes of 0.2N NaOH, 1% SDS solution, mix carefully, and leave at room temperature for 5 minutes. Add 0.5 volume of 5M potassium acetate solution to the reaction solution After 5 minutes, the mixture was centrifuged at 12,000 g for 10 minutes to obtain a supernatant. Extract the supernatant 2-3 times with phenol and phenol / chloroform / isoamyl alcohol (25: 24: 1), add 0.1 volume of 3M sodium acetate and 2 volumes of ethanol. Plasmid DNA was precipitated. The precipitated DNA was again dissolved in TE buffer (10 mM Tris-Cl, pH 8.0 / 1 mM EDTA, pH 8.0) and CsCl was added to 1 g / ml. The solution was centrifuged at 100,000 rpm for 12 hours using a Backman TL 100 roter to purify the plasmid DNA. A band containing plasmid DNA was separated by syringe, extracted with n-butanol, and dialyzed in TE buffer for 24 hours. Plasmid DNA was extracted, digested with EcoRI and ClaI restriction enzymes, and electrophoresed on 0.8% agarose gel to produce human papillomavirus type 16 E6 / E7 gene of 3.52 Kb (hereinafter referred to as "TYR-HPV16E6E7") (Figure 1). Agarose fragments containing DNA were cut out to purify DNA. Tyrosinase minigene (Fig. 1) of 4.5 Kb was purified by electrophoresis on agarose gel in the same manner as above. After quantifying the DNA concentrations of the two purified gene fragments, the mixtures were diluted with 10 mM Tris-Cl, pH 7.5 / 0.2 mM EDTA, and pH 8.0 buffer so that the DNA concentration was 4 ng / μl, and then -20 ° C. Store in and use for microinjection.

Step 2: Microinjection of Human Papilloma and Tyrosinase Expression Genes

Females over 6 weeks old from F & B mice purchased from B & K (UK) were selected for superovulation to obtain a large number of fertilized eggs from a small number of donor mice. The best time to inject DNA into the male pronucleus of fertilized eggs is the single-cell stage of mouse fertilized eggs, depending on the type of mouse of the supplier and the light-dark cycle of the animal compartment. . FVB pure mice were used and microinjection usually started at 10:30 am by adjusting the light-dark cycle of the animal room to be turned off at 7 pm and turned on at 6 am. One fertilized egg (egg) was fixed at a position where the male pronucleus was easily seen, and 1-2 pl of DNA was injected into the male pronucleus with an injection pipette.

Step 3: Insertion of Microinjected Embryos into Surrogate Mouse Oviducts

Among the fertilized eggs inserted with DNA, healthy and lysed were sorted and discarded, and the healthy ones were transferred to foster mother mice that day or put in M16 medium and incubated in a 37 ° C incubator. Male mice mating with surrogate mothers used ICR, and when they were mated with other female mice after the vas deferens, it was confirmed that the pups were not born even with vaginal plugs. ICR female mice close to the ovulatory phase were used as surrogate mothers if there was a vaginal plug to be mated with the male mice undergoing intubation. Surgery was performed by intraperitoneal injection of 0.2 ml of anesthetic with 10x dilution of sonnopentyl (64.8 mg sodium pentobarbital / ml from Pitman-Moore Co. After insertion into the oviduct, the surgical site was closed.

Step 4: Breeding of Transgenic Mice

Transgenic mice that were confirmed to have inserted human papillomavirus genes by PCR of the tail DNA were crossed with normal FVB pure mice after 6 weeks of age. In the morning, vaginal plugs were examined to check for fertilization. If normal mating was not performed, normal mice were exchanged to check for fertilization. When the baby (F1) was born, the hair color was first visually identified to distinguish black and white, and after 3-4 weeks, PCR was performed using tail DNA to examine how human papillomavirus genes were transferred in relation to the groping. . When the groping was confirmed that the human papilloma virus gene was delivered, it was named as a transgenic line. F1 transgenic mice were again mated with normal FVB transcript mice 6 weeks after birth.

On the other hand, in order to make homozygous transgenic mice, mating was performed between sisters from the same surrogate mother mouse among F1 transgenic mice. In order to find homozygous mice among the F2 transgenic mice born between them, they were crossed again with normal FVB scavenger mice to obtain F3 mice, and then groping and human papilloma virus genes were examined.

Example 2. Characterization of Human Papilloma Virus Transgenic Mice

Step 1: extract tail DNA

The polymer DNA was extracted from the tail of the mouse and analyzed by PCR to determine the presence of the transgene.

After cutting the tail of the mouse about 3-4 weeks, cut it 1.5cm, and then finely cut it into 500 μl of 50 mM Tris-Cl, pH 8.0 / 50 mM EDTA, pH 8.0 / 0.5% SDS buffer, and proteinase K (proteinase K). ) Was added to 200 μg / ml and reacted at 55 ° C. for 9 hours. This was extracted three times with the same amount of phenol and phenol / chloroform / isoamyl alcohol (25: 24: 1) and dialyzed for 36 hours (12 hours × 3 times) with 10 mM Tris-Cl, pH 8.0 / 1 mM EDTA, pH 8.0 solution. It was. The concentration of DNA was measured with a spectrophotometer and the DNA solution was stored at 4 ° C.

Step 2: PCR analysis

Using PCR tubes purchased from KOREA, 2 μL of each sample DNA, 1 μL of each of the sense and antisense primers were added thereto, and the mixture was filled with 50 μl of sterile distilled water and mixed. At this time, the primer is present in the region shown by the arrow of Figure 1 and the base sequence of the primer is disclosed in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. Mineral oil was added to the upper layer of the mixed solution and heat-treated at 95 ° C. for 5 minutes to sufficiently denature the DNA, followed by temperature change at 72 ° C. Thereafter, PCR cycles of 1 minute (denaturation) at 94 ° C, 1 minute (annealing) at 55 ° C, and 1 minute (extending) at 72 ° C were performed 30 times. After the appropriate PCR cycle was completed, the reaction mixture was extended for 5 minutes to induce the amplified DNA fragments to sufficiently form a double helix, and then, 1-2 µl of these were electrophoresed to determine the result.

Figure 2 shows the experimental results, P represents a positive control group, M represents a DNA molecular weight marker, the human papilloma virus gene was transplanted into the mice because the same band as the positive control in transgenic mice 6, 7, Able to know. In addition, all of the mice transplanted with the gene were found black from the beginning of the hair color, and afterwards, it can be seen that the graft can be easily visually determined without a special genetic test.

Stage 3: Histopathological examination of the back skin

A 5 μm section of the dorsal skin of 5-6 month old transgenic mice was fixed in 10% paraformaldehyde, embedded in paraffin, and stained with hematoxylin and eosin through a conventional procedure. The result is a shooting (100x magnification). The epidermal layer of the skin was overproliferated in all four histopathological examinations, indicating that the expression of the transplanted human papillomavirus type 16 E6 / E7 gene causes skin diseases.

Step 4: Western blot analysis of each tissue of transgenic mouse

Brain, heart, eye, kidney, skin, spleen, lung, testis and liver tissues of 2-3 months old F1 transgenic mice to determine where the gene is expressed in mouse tissues in which human papilloma virus has been transplanted A portion of the was removed and the incision was well cut with scissors. The dissected tissue was placed in a tube, added SDS gel loading buffer, and then soaked in boiled water for 10 minutes. Boiled tissue solution was centrifuged at 10,000g for 10 minutes and the supernatant was taken. The extracted protein was quantified and separated in 10% SDS-PAGE at a concentration of 100 µg, and then transferred to a nitrocellulose paper using a semiphore transfer unit (Hoeffer). The transferred nitrocellulose paper was washed three times with PBS three times for 10 minutes and blocked with 5% skim milk / PBS for 2 hours at room temperature and washed again with PBS. Subsequently, the test for the E6 / E7 expression protein was diluted 1: 1000 with the monoclonal antibodies HPV-TVG710Y and HPV-C1P5 purchased from Santa Cruz Biotech. And reacted overnight at 4 ° C. The reacted nitrocellulose paper was washed again with PBS, and then diluted with anti-mouse immunoglobulin G (anti-mouse IgG) labeled with Peroxidase 1: 1000 and reacted at room temperature for 2 hours, followed by 4-chloro- 1 naphthol (4-chloro-1-naphtol) and hydrogen peroxide were added to give a color reaction.

Figure 4 shows this result, Br is brain, Ey is eye, He is heart, Ki is kidney, Sk is skin, Sp is spleen, Lu is lung, Te is testis, Li is liver tissue. It can be seen that the human papilloma virus type 16 E6 / E7 protein is expressed in the heart. Therefore, it can be seen that the pathological histological cause of hyperplasia of skin tissue is confirmed once again.

Stage 5: Two-stage cancer model test

In order to confirm the availability of the transgenic mice prepared previously, skin cancer-causing tests were performed in transgenic lines of human papillomavirus-transplanted mice and normal FVB-based mice. All animals to be used for the test were clipped using a clipper 2 days prior to drug treatment. 7,12-dimethylbenzanthracene (7,12-Dimethylbenz [a] anthracene) (DMBA) was dissolved in acetone at a concentration of 25 µg / 200 µl and applied to the precut area. One week after DMBA application, 12-O-tetradecanoylphorbol-13-acetate (TPA) was dissolved in acetone at a concentration of 2.5 μg / 200 μl, twice a week. Applied to the site. TPA application was carried out for 15 weeks, and the applied liquid amount was 200 μl per horse. The clipper was used to shear the hair until the skin papilloma developed during the application was not affected. Skin papilloma development was observed at the time of application, and the time of initial onset and the number of skin papillomas over 2 mm in diameter were counted and recorded. The inspection result is shown in FIG. Mice transplanted with human papillomavirus developed dermal papilloma at a faster time than normal mice and had a higher frequency. Particularly, in females, 15 weeks after the test treatment, the incidence of dermal papilloma in transgenic mice was 69.2%, which is about 2 times higher than that of normal mice. Therefore, it can be seen that the incidence of skin diseases caused by human papilloma virus is increased. Figure 6 is a photograph of the skin papilloma-induced aspects of transgenic mice and normal mice.

As described above, transgenic mice transplanted with the human papillomavirus type 16 E6 / E7 gene of the present invention had a natural skin hyperplasia and a skin papilloma incidence (69.2%) during the skin cancer induction test (30%). ), Which is more than twice as high as that of), can be usefully used in research fields such as identifying mechanisms of development of diseases related to human papilloma virus type 16, developing therapeutic agents, and new drug assay systems.

Claims (10)

Human papilloma virus 16 E6 / with germ cells and somatic cells having a recombinant expression vector comprising a human papilloma virus type 16 E6 / E7 gene linked to a promoter for expressing the human papilloma virus type 16 E6 / E7 gene. E7 transgenic mice. The human papilloma virus type 16 E6 / E7 gene transplant mouse according to claim 1, wherein the promoter for expressing the human papilloma virus type 16 E6 / E7 gene is a tyrosinase promoter. The human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 1 or 2, wherein the recombinant expression vector is TYR-HPV16E6E7. The human papillomavirus type 16 E6 / E7 transgenic mouse according to claim 1 or 2, further comprising a tyrosinase minizin in said germ cells and somatic cells together with said recombinant expression vector. The human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 4, wherein the recombinant expression vector is TYR-HPV16E6E7. A recombinant expression vector comprising a human papilloma virus type 16 E6 / E7 gene linked to a promoter for expressing a human papilloma virus type 16 E6 / E7 gene was prepared, and the recombinant expression vector was introduced into a fertilized egg of a mouse. A method for producing human papilloma virus type 16 E6 / E7 transgenic mice comprising culturing healthy fertilized eggs and inserting them into the oviduct of surrogate mother mice. The method for producing a human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 6, wherein the promoter for expressing human papilloma virus type 16 E6 / E7 gene is a tyrosinase promoter. The method of manufacturing a human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 6 or 7, wherein the recombinant expression vector is TYR-HPV16E6E7. The method for producing a human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 6 or 7, wherein a tyrosinase minigene is further introduced into the fertilized egg simultaneously with said recombinant expression vector. The method of manufacturing a human papilloma virus type 16 E6 / E7 transgenic mouse according to claim 9, wherein the recombinant expression vector is TYR-HPV16E6E7.