Feasibility study of RCAS-TVA transduction system in the model of mouse liver

• Main Authors: Wu Yu-lien, 吳鈺廉
• Other Authors: Tzeng Yin-jeh
• Format: Others
• Language: zh-TW
• Online Access: http://ndltd.ncl.edu.tw/handle/89662494765266323489

碩士 === 慈濟大學 === 分子生物及細胞生物研究所 === 91 === The purpose of this research is to evaluate the in vivo gene transfer method with RCAS-TVA transduction system in the mouse liver. This system, developed in Harold Varmus’s laboratory, is a combination of avian retroviral gene transduction system and transgenic mice technology. The principle of this system is based on the introduction of the avian retroviral receptor TVA (receptor for subgroup A avian leukosis virus) into mice, which permits the infection of mouse cells by the avian retroviral vector, RCAS (Replication-Competent, ALV-LTR, Splice acceptor) carrying various genes of interested. ALSV (avian leukosis virus subgroup A) is similar to other conventional retroviruses, can not infect nondividing cells. With the intention a new system is developed in Harold Varmus’s laboratory. They have developed a system for the preparation of a human immunodeficiency virus type 1 (HIV-1) based lentiviral vector, pseudotyped with the envelope protein of ALSV subgroup A. The HIV(ALSV-A) vector retains the requirement for TVA on the surface of target cells. This system can introduce genes into somatic cells in TVA transgenic animals and allow evaluation of the effects of altered gene expression in differentiated cell types in vivo. Hepatitis B virus (HBV) is closely linked to hepatocellular carcinoma (HCC) in Taiwan. In this thesis we intend to study the HBV gene HBx and HBy with RCAS-TVA system. HBx is a pleiotropic transactivator of viral promoter and enhancer. HBy is a putative gene found by our laboratory of which the encoding nucleotide is antisense to HBx. In order to successfully infect mouse liver with ALSV-based vector, transgenic mice carrying TVA under the control of liver-specific promoter albumin (AB) or phosphoenolpyruvate carboxykinase (PEPCK) were generated in this laboratory. By now we have generated two lines of AB-TVA and six lines of PEPCK-TVA transgenic mice. Based on the RNA analysis showed five lines of transgenic mice expressing TVA receptor have been determined. The GFP-RCAS virus was generated by DF-1 cell with RCAS-GFP plasmid, and injected to hepatectomized mice 24 and 48 hours after partial hepatectomy (PHx). Seven days later we collected the mice liver for Western blot anaylsis, paraffin embedding, and frozen tissue section. By immunohistochemistry and fluorescence microscopy, we have detected the GFP expression in frozen sections. The molecular analysis from these observations, we concluded that the RCAS-TVA system can transduce foreign gene into the mouse livers. Furthermore detection of GFP expression by means of western blotting anaylsis and fluorescence microscopy showed that the HIV(ALSV-A) system is able to transduce foreign gene into nondividing mouse liver cells in vivo.