Stable Transmission and Expression of the Hepatitis B Virus Total Genome in Hybrid Transgenic Mice Until F10 Generation HAYDAR BAGIS 1Ã , SEZEN ARAT 1 , HANDE ODAMAN MERCAN 1 , DIGDEM AKTOPRAKLIGIL 2 , MUGE CANER 2 , EDA TAHIR TURANLI 1,3 KEMAL BAYSAL 1 , GAZI TURGUT 1 , SAKIR SEKMEN 1 , AND BEYAZIT CIRAKOGLU 1,2 1 TUBITAK, Research Institute for Genetic Engineering and Biotechnology (RIGEB), Transgenic Core Facility, Kocaeli, Turkey 2 Department of Medical Biology, School of Medicine, Marmara University, 34668 Istanbul, Turkey 3 Istanbul Technical University, Department of Molecular Biology and Genetics, Maslak, 34469 Istanbul, Turkey ABSTRACT The aim of the present study was the generation of transgenic mice carrying the complete Hepatitis B Virus (HBV) genome and investigation of the presence of Hepatitis B surface antigen (HBsAg) expression through successive generations. Transgenic mice were generated by microinjecting HBV genome into fertilized eggs. Integration and expression of HBsAg in transgenic mice were analyzed by genomic DNA PCR, Southern and slot blots and enzyme-linked immunosorbent assay (ELISA). Expression was also confirmed by Western blotting and RT-PCR. Histological changes in liver tissue of transgenic mice were examined by HE staining. The HBV genome was transmitted to the F10 generation and the presence of HBV X gene transcripts was confirmed by RT-PCR analysis using liver cDNAs from the F10 generation mice. During an observation period of 2.5 years, mice were sacrificied and their organs subjected to histopathological examination. In the liver, slight histopathologic alterations were observed but none of these lineages had any hepatocellular carcinoma (HCC). HBV DNA can be stably transmitted and expressed in the transgenic mice until F10 generation. However, although we showed the presence of X gene transcripts in liver tissues of F10 generation mice by RT-PCR in these animals, long-term expression of the HBV complete genome and expression of X protein in hepatocytes did not cause neoplasia during the life span and HCC. These transgenic mice should be useful for detailed studies of the replication and expression of HBV and for physiological studies of HBV genome. J. Exp. Zool. 305A:420–427 2006. r 2006 Wiley-Liss, Inc. Hepatitis B Virus (HBV) genome has four open reading frames, including envelope genes coding region (pre-s1, pre-s2 and s gene coding region), precore (pc) and core(c) gene coding region, polymerase (p) gene coding region and x gene coding region (Chisari, ’95; Chisari and Ferrari, ’95). The HBV is a noncytopathic envel- oped virus with a small (3.2 kb) circular double- stranded DNA genome. HBV causes various liver diseases such as acute to chronic hepatitis, cirrhosis and hepatocellular carcinoma (HCC) (Chisari, ’95; Chisari and Ferrari, ’95). However, it is unclear which properties of the virus are responsible for these long-term consequences. It is generally accepted that HBV is not directly cytopathic and it is proposed that the acquisi- tion, maintenance and outcome of chronic carrier- ship are mainly dependent on the immune status of the host (Peters et al., ’91). Cell culture systems have been established that allow expression and replication of the HBV genome following transfection with cloned HBV DNA (Sureau et al., ’86; Yaginuma et al., ’87). These systems have allowed detailed molecular and genetic Published online 17 February 2006 in Wiley InterScience (www. interscience.wiley.com). DOI: 10.1002/jez.a.277. Received 15 August 2005; Accepted 8 December 2005 Grant sponsor: Turkish Scientific and Technical Research Council ProjectSBAG MAM1 102 S 005; Grant sponsor: Foundation of Marmara University. Ã Correspondence to: Assoc. Prof. H. Bagis, TUBITAK-GMBAE, Transgenic Core Facility, P.O. Box 21 41470, Gebze, Kocaeli, Turkey. E-mail: haydar@gmbae.tubitak.gov.tr r 2006 WILEY-LISS, INC. JOURNAL OF EXPERIMENTAL ZOOLOGY 305A:420–427 (2006)