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【摘要】 It is vital to build an ideal animal model in studying diseases. This is also true of the case of hepatitis B. It is not only for the study of the infection or pathogenic mechanism of hepatitis B, but for the screening of anti-HBV agents. Transgenic mouse has been featured as the most idealistic model organisms. This literature review focuses on the methods of establishing transgenic mice model of hepatitis B virus in human and compares their characteristics.
【关键词】 Hepatitis B virus Animal model Transgenic technique Mouse
Hepatitis B is known to be one of the pandemic infectious diseases. The infection of hepatitis B virus (HBV) has become one of the serious global public health problems. There have been many studies on the pathogenesis and mechanism of hepatitis B in recent years.
However, as the number of the hosts of HBV is very small and only humans and some other primates are vulnerable to this virus, there has been a big obstacle in studying the disease, since it was difficult to establish animal models for hepatitis B.
However, since transgenic technology was invented, the human can study any interesting genes through the development of transgenic animals. The establishment of transgenic mice becomes an effective method of studying hepatitis B.
1 Transgenic animal technology and development of HBV transgenic animals
1.1 Transgenic animal technology In transgenic animal technology, exogenous genes are introduced artificially into protein-coding genes of other animals. It can be integrated into the gene of the animal and it will be later expressed. This is a kind of biotechnology of studying target gene. Its characteristic is that an operation at the molecular and cellular level can result in an expression at organizational and overall levels. The main methods of making transgenic animals are microinjection, progenitor cells transplantation technology, retrovirus as a vector, embryonic stem cells as a vector, adenovirus carrier and sperm carrier. Transgenic Animal Technology has existed since the 1980s, with the in-depth study. The technology has brought breakthrough developments. Prokaryotic microscopic injection is the first application of the method. Since then, transgenic animal technology, such as the embryonic stem cells, retrovirus infection, somatic cell nuclear transfer method and artificial yeast chromosome method has been the best method［1］.
1.2 Transgenic animals A transgenic animal of hepatitis B gets exogenous HBV DNA fragments fully or partially integrated into its genome. In addition to the current several rare primates, which have been experimentally infected, there is no ideal experimental animal. However, since the emergence of transgenic technology, people have built transgenic mice. In August 2002, the genome map of mouse was formally published and it became clear that people and mouse share 80 percent of the genetic materials and 99 percent of the gene. Therefore, the researchers came to believe that the understandings of genetic information of mouse will lead to human research. Genome draft showed the fact that about 2.5 billion base pairs on the 20 pairs of chromosomes in mouse are very close to 2.9 billion base pairs on the 23 pairs of human chromosomes, DNA chain between genes and gene fragments are very similar. And mice also have some characteristics of small individual, low cost, strong reproductive capacity, short reproductive cycle, and high efficiency of genetic transformation［2］. It is more idealistic than any other animals which were taken into consideration.
2 Preparations for transgenic mouse of Hepatitis B
After Chisari successfully constructed the first transgenic mouse containing HBV gene in 1995, many laboratories in the world also followed his success and developed transgenic mouse expressing different HBV genes. The processes of preparing transgenic mouse of hepatitis B are divided into the following categories.
2.1 Microscopic prokaryotic injection This method was firstly established by Gordon in 1980 and it has been the most popular method. It is reliable and relatively stable. The basic process is as followed; first, foreign genes are injected directly into fertilized eggs of laboratory animals by using microscopic operating system and microinjection technology. Next, the fertilized eggs with the foreign genes inserted are transplanted into the uterus of the donor animals through the embryo transfer technology. And we need to wait till it turns into transgenic animals, the majority of which can take integrated genes to future generations in the course of experiment and create transgenic animals system.
2.1.1 Transgenic mouse with some fragments of HBV In 1990 and 1991, Lee［5］ and Kim［6］ established transgenic mouse model with HBx by microscopic prokaryotic injection. TU Ya-jun［7］ injected an 1800 bp-fragment gene of hepatitis B virus X proteins (HBx) into fertilized eggs of Kunming mice and successfully established mouse models with X gene in adr subtype. SU Xiao-ping［8］ constructed transgenic mouse of expressing preS2-S gene of hepatitis B virus (HBV, adr subtype) in C57BL / 6 mice. Subsequently, LIU Hong［9］ constructed the eukaryotic expression vector pcDNA3-HBc and then injected its subsequent use of this linear technology into the C57BL/6 mouse embryos, so as to establish transgenic mouse with core gene of HBV.
2.1.2 Transgenic mouse with full-length sequence of HBV LEI Zhang-heng used an ICR mouse and constructed transgenic mouse model with ayw subtype of HBV through microscopic prokaryotic injecting［10］. HU Wei-jiang also constructed transgenic mouse model containing 2 copies of hepatitis B virus (HBV, adr subtype) by using C57 mouse［11］.
2.1.3 Transgenic mouse with super-long sequence of HBV In 1995, Guidotti build a 4096bp-long sequence of HBV by inserting repeated C genes and X genes and enhancer in 5' end of the full-length sequence of HBV. He then took them into embryos of mouse: this was the first transgenic mouse with super-long sequence of HBV［12］. Wu Jin-ming transformed plasmid (PT-HBV1.3) into DH5α(E. coli) and digested it with Hind III and Sac I. He received a fragment with 4.1kb, which was equal to 1.3 times the length of the fragment of HBV. He injected the super-long fragment into zygote of Kunming mouse and finally, he succeeded in building transgenic mice with super-long sequence of HBV［13］.
At present, microscopic prokaryotic injection is still the most commonly used transgenic method, and it has provided successes. However, there are some shortcomings in its application: First of all, the method is expensive and it has low rate of integration. This method is time-consuming and its operation is complicated. It needs expensive laboratory equipment, and the integration rate is as low as 1 percent of the injected eggs. Second, the method has great randomness of integration and we do not have the ability of site-specific integration. When prokaryotic was injected into exogenous gene, it can be inserted into any site of the genome chromosome receptor. The genetic expression and stability are not guaranteed［14～16］.
2.2 Retrovirus as a vector In 1974, Jaenisch utilized retrovirus as a vector of HBV to infect embryos. The basic process is listed below: First, they recombined target gene with retrovirus and made virus particles of high titer. And then, they infected the embryo of pre-implantation or implantation with the retrovirus. The virus with foreign gene was inserted. And the aim of infection was received.
DING Chuan-lin had infected the bone marrow progenitor cells of mouse in its adivision stage with recombinant of retroviral vector containing C gene of HBV［17］. He successfully integrated target gene into the genomic DNA of dendritic cell. It began transcription and translation. ZHANG Zheng also injected mice with the mixture of antisense RNA retrovirus vector containing X, P dual-target district of HBV and liposome. He successfully integrated the retroviral vector into the liver cells of mouse by utilizing the characteristic of the liposomes that they can be easily traced［18］.
One of the advantages of this method is that it introduces single-copy gene and it does not destroy the target gene or cause a major mutation. In addition, it is easy to analyze inserted site. Its disadvantages are that the inserted gene is very short, that the test needs the chimeric way and that it has a long cycle. Moreover, there may be diverted to the replication of the virus［19］.
2.3 Spermatogonial stem cells mediated Spermatogonial stem cells (SSCs) have high self-renewal capacity and high differentiation potentials. They are diploid immortal cells, which can be replicated only in male. They are transferred to the next generation. The principle of this method is that the gene of HBV is directly injected into the seminiferous tubules of male mice and then spermatogonial stem cells are infected.
HU Huo-zhen first constructed an expression vector (pc-DNA3-X) with the needle of Microscopic injection. The liposome of the vector was with coated X gene of HBV and injected it into the seminiferous tubules of male mice (C57BL/6). Thereby, the transgenic mice with X gene of HBV were successfully built［20］. LIUGuang-ze took the gene of HBV (adr subtype) as target genes and cloned it to PBR322 plasmid, and mixed with liposomes according to a certain proportion. At last, he succeeded in transfecting the SSCs of Kunming mice［21］.
The advantages of this method are: ① it overcomes the shortcomings of random integration in the microstructure prokaryotic injection, for it is possible to insert exogenous gene into the particular location of chromosome, or to have gene mutate and to achieve fixed-point integration of exogenous gene. ② Gene targeting or gene knockout can produce the animal with a gene deletion, which is a new approach of establishing certain diseases model. However, low efficiency of reorganization is the biggest weaknesses of this method.
2.4 Sperm carrier Sperm-mediated gene transfer (SMGT) is a method in which sperm is used as a carrier. Exogenous gene is inserted into oocytes at the fertilization. This is one of the most simple and efficient methods in transgenic animals study. Through the development of the sperm-mediated gene transfer technology over the last ten years, the researchers have used sperm as the carrier of the exogenous genes and integrated them within animal embryos in the fertilization process, so that its offspring has foreign genes.
XIONG Xiao-fang has firstly transformed pBR322 of HBV into competent cells DH5α. He plated and picked colonies of positive clone after screening in ampicillin. He, then cultured, extracted the products and obtained purified plasmid. He transduced directly pBR322-HBV transfection liquid into the testicular tissue of healthy mature male mice, and at last it was integrated into the oocyte through natural insemination［22］.
The method has the following advantages. ① It is simple, easy to operate, and costs little. The operation does not require expensive facilities or complex skills. ② It can obtain fertilized eggs without sacrificing rats.
But it has very low rate of integration and the integration of DNA and sperm is vulnerable to a number of factors. There are unstable results because the experimental conditions can be easily affected.
3 Prospects
At present, there are transgenic mouse model, duck model, immunodeficiency mouse model of infection, tree shrews model and primate model such as chimpanzees as main animal models of hepatitis B［23］. Although the pathogenesis of duck of HBV is similar to that of human, the fact that ducks lack X gene in HBV is critical because the ducks carrying the virus does not develop cirrhosis or hepatocellular carcinoma［24］. At this point, it is different from the natural process of human HBV infection. Immunodeficiency mouse model of Hepatitis B does not apply to the study of immune clearance mechanisms, tree shrews and primates are rare and not accessible. Therefore, the use of the transgenic mouse has been widely adopted as an economic, convenient and simple animal experiment of hepatitis B.
In summary, the methods of constructing transgenic mouse model of hepatitis B exists microscopic prokaryotic injection, retrovirus as a vector, spermatogonial stem cells mediated and sperm carrier and microscopic prokaryotic injection is the oldest, most wide, and most mature of transgenic technologies. But retrovirus as a vector, spermatogonial stem cells as a vector and sperm carrier provides a new way for constructing the transgenic mouse model of hepatitis B. The specific method used in the experiment should be based on the purpose and conditions of the laboratories. We believe that transgenic mouse model of hepatitis B will become the ideal animal model of human hepatitis B to study the pathogenesis and prevention, particularly to screen drugs and test vaccines with the improvement of theory and technology. It will have a huge contribution to the treatment of hepatitis B.
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作者单位：Department of microbiology & immunology, DALI University, Yunan, Dali 671000, P. R. China.