The Establishment Of Transgenic Mice Carrying 1.3copy Hepatitis B Virus Genome(C Subtype)

Hepatitis B virus (HBV) is a small DNA virus belonging to hepadnavirus family. HBV can cause the acute and chronic hepatitis and also hepatocellular carcinoma (HCC). At present, eight genotypes (A-H) can be distinguished by its HBV sequence difference. The eight genotypes of HBV have a distinct geographic distribution. In China, genotype A, B, C and D have been found, of which genotype B and C are the the most prevalent genotypes. Genotype C is epidemic all over our country and the most prevailing virus strain in Northern China,,and is even up to 69% in some city of North China.Under natural conditions, HBV can only infect primates such as chimpanzee and can not infect the ordinary rodents such as mice. Owing to a narrow range of hosts, These has greatly confined the research of hepatitis. With the development of transgenic technology, several mouse strains carrying 1.0 or 2.0 copy HBV transgenes have been achieved, which offered opportunity to investigate the biology of HBV, the pathogenetic mechanisms responsible for HBV associated liver diseases and development of anti-HBV vaccine and drug treatment. While HBV can replicate in the murine hepatocyte of HBV transgenic mice, further experiments are greatly limited because they replicate the virus at very low levels. Both study with cell model and transgenic mice indicates that 1.3 copy genome construct can drive HBV expression and replication at the levels comparable to that in naturally infected liver. But presently the HBV transgenic mice (genotype C) are available at home and abroad. Therefore, our group contructed 1.3 copy C genotype fragment firstly in the world. This study is to generate transgenic mice carrying 1.3copy HBV (C) genome to provide with more suitable animal model for the related researches in China.In the present study, we used the vector of pUC19-HBV(C)1.3 containing the 1.3 copy HBV DNA fragment. This plasmid was identified by restriction endonucleases digestion, followed by confirming by sequencing. The sequence of 1.3 copy HBV was coincident with the reported 1.3 copy HBV sequence. The identities between the 1.0 copy of .3 copy HBV DNA and the canonical DNA sequence of C genotype reported, was up to 98%. This identified the genotype of this 1.3 copy HBV was C genotype prevailing in China.The extraction of plasmid was operated to yield DNA fragment used for microinjection to produced to transgenic mice. The objective DNA fragment was recovered by comparison with the staining gel.DNA solution was diluted to 3ng/ul. 4-5w FVB/N females were used as embryo donors, Females was superovulated by intro-peritoneal(i.p.) injection of 5IU pregnant mare serum gonadotropin, followed by i.p. injection of 5IU human chorionic gonadotropin 48hr later, then placed individually with stud males. Pseudopregnancy KM females were choosed as embryo receivers, Pseudopregnancy females were characterized by having plugs, engorge and rosy valva, which was made by mating with aciesis male of deferent duct deligated.The purified DNA fragments were injected into the pronucleus of FVB/N zygotes. The survival zygote after microinjection were reimplanted into the oviducts of psudopregnant KM females. About 21 days after implantation, the pups were born, followed by identifying the integration, its replication and expression.of foreign gene,①3 weeks after birth,, genomic DNA was extracted from the tail tips to check if transgene were integrated into the mouse genome, and then screen positive transgenic animal.②Getting blood from vena orbitalis posterior, centrifugated blood serum were used for serum HBsAg/HBeAg ELISA and HBV DNA RT-PCR detection.③The intracellular distribution of HBsAg and HBcAg in liver were assessed by Immunohistochemical analysis of liver section.In this study, 2840 zygotes were injected, the survival 2256 zygotes after microinjection were reimplanted into the oviducts of 85 psudopregnant KM female mice. After birth, 42 female mice were pregnant and 168 pups were born. PCR analysis indicated that 14 out of 168 pups were integrated with HBV genome. With ELISA, 5 of them express HBsAg in serum. And two mice were detected HBV DNA replication in mice serum with number of 3.12×10~2copy/ml, 1.98×10~2copy/ml repectively.Then the transgenic mice were mated to normal mice to establish transgenic mouse strain. 61 F2 offsprings were born. Further study demonstrated that 5 of them were integrated with HBV genome by PCR ,4 of them serum HBsAg positive by ELISA and 2 of them with the intracellular distribution of HBsAg in hepatocyte by immunohistochemistry. Both serum HBeAg and liver HBcAg immunohistochemistry were negative.These results suggest that HBV transgene could be inherited to the next generations, replicate and express in transgenic mice. This transgenic mouse model is useful for investigating the pathogenetic mechanisms responsible for HBV associated liver diseases and development of anti-HBV vaccine and drug treatment, especially for the study of C genotype strain prevailing in China.