Analysis of Pathogenesis of Chimera Mice Introduced with the Immediate Early Genes of Murine Cytomegalovirus

尽管我们尽了最大的努力来获得项目的称号，但我们还是不能完成项目的最终目标。然而，在这方面，本研究通过几个步骤实现了小鼠巨细胞病毒（MCMV）即早期（IE）基因的克隆，并将其作为BamH Ⅰ片段克隆到pACYC 184质粒载体中，比较了将MCMV即早期（IE）基因与表达载体连接后转染不同培养细胞的表达效率通过电穿孔法将IE基因导入胚胎干细胞（ES-D3细胞）中，并在G418存在下用免疫荧光法筛选表达IE基因的克隆细胞。4）将克隆的DE细胞导入BDF 1小鼠的囊胚中，并将其回输于假孕ICR小鼠的子宫中，制备嵌合小鼠。虽然我们将约100个注射ES细胞的囊胚回输到10只ICR小鼠的子宫中，但我们通过PCR扩增在两个后代的尾部DNA中没有检测到IE基因。5）我们还尝试使用连接IE基因启动子和lacZ基因的DNA构建体来制备转基因小鼠。将构建体注入受精卵后，我们从36只后代中获得了4只转基因小鼠。我们现在正在研究lacZ基因的表达。

Altough we have made best effort to attain the title of projicts, we could not accomplish the final goal of the project. However, we have overcome several steps to approach the goal and developed the techniques for it.1) We have done the clonning of the immediate early (IE) genes of murine cytomegalovirus (MCMV) into the pACYC184 plasmid vector as a Bam Hl fragment.2) We have compared the efficiencies of the IE gene expression after conection of the gene to the expression vector and transfection into various cultured cells.3) The IE genes were introduced into the embryonic stem cells (ES-D3 cells) by electroporation of the gene and the cloned cells which expressed the IE gene were selected in the presence of G418 by assying using immunofluorescence.4) We have tried to make chimera mice by inroducing the cloned DE cells into the blastocysts of BDF1 mice and returned in the uteri of pseudopregnant ICR mice. Although we returned the about 100 of the ES cells-injected blastocysts into the uteri of 10 ICR mice, we could not detect the IE gene in the tail DNA of two offspring by PCR amplification.5) We have also tried to make the transgenic mice using the DNA construct which connect the promoter of the IE gene and lacZ gene. We have gotton 4 transgenic mice out of 36 offspring after injection the construct into the ferilized eggs. We are now under way to examine the expression of the lacZ gene.