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

鼠巨细胞病毒早期基因导入嵌合小鼠发病机制分析

• 批准号: 04670222
• 负责人: TSUTSUI Yoshihiro
• 金额: $ 0.9万
• 依托单位: Institute for Developmental Research, Aichi Parefectural Colony
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1992
• 资助国家: 日本
• 起止时间: 1992 至 1993
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-04670222/
• 关键词: cytomegalovirus; chimera mice; gene expression

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.

尽管我们尽了最大的努力获得项目的称号，但我们没有达到项目的最终目标。然而，为了达到这一目标，我们克服了几个步骤，并开发了相应的技术。1)我们将小鼠巨细胞病毒(MCMV)的即刻早期(IE)基因克隆到pACYC184载体中，作为BamH1片段。2)我们比较了将IE基因连接到表达载体并导入不同培养细胞后的表达效率。3)通过电穿孔将IE基因导入胚胎干细胞(ES-D3细胞)，并在G418存在下用免疫荧光法筛选到了表达IE基因的克隆细胞。4)将克隆的DE细胞导入BDF1小鼠的囊胚，并返回假孕ICR小鼠的子宫内，建立嵌合体小鼠。虽然我们将约100枚胚胎细胞注射的囊胚送入10只ICR小鼠的子宫内，但在两只后代的尾部DNA中均未检测到IE基因。5)我们还尝试用连接IE基因启动子和LacZ基因的DNA构建了转基因小鼠。将构建好的转基因小鼠注射到受精卵中，36只后代中获得了4只转基因小鼠。我们现在正在检测LacZ基因的表达。

项目成果

柏井明子,河村則子,門田智佳,筒井祥博: "Susceptibility of mouse embryo to murine cytomegalovirus infection in early and mid-gestation stages" Archives of Virology. 127. 37-48 (1992)

Akiko Kashiwai、Noriko Kawamura、Tomoka Kadota、Yoshihiro Tsutsui：“妊娠早期和中期小鼠胚胎对鼠巨细胞病毒感染的敏感性”病毒学档案 127. 37-48 (1992)。

筒井祥博,柏井明子,河村則子,門田智佳: "Microphthalmia and cercbroal atrply induced in mouse embryos by infection with murine cytomegalovirus in midgesfation." American Journal of Pathology. 143. 804-812 (1993)

Yoshihiro Tsutsui、Akiko Kashiwai、Noriko Kawamura、Tomoka Kadota：“妊娠中期感染小鼠巨细胞病毒诱发小鼠胚胎小眼症和脑动脉瘤。”143. 804-812 (1993)

門田 智佳,長浜 真人,筒井 祥博: "Reletionship between HOXZ homeobox gene expression and the human cytωlovirus immediate early genes." Journal of General Virology. 73. 975-981 (1992)

Tomoka Kadota、Masato Nagahama 和 Yoshihiro Tsutsui：“HOXZ 同源盒基因表达与人类细胞病毒直接早期基因之间的关系。普通病毒学杂志”73. 975-981 (1992)。

Tsutsui Y, Kashiwai A, kawamura N, and Kadota C: "Microphthalmia and cerebral atrophy induced in mouse embryos by infection withmurine cytomegalovirus in midgestation." Am.J.Pathol.143. 804-812 (1993)

Tsutsui Y、Kashiwai A、kawamura N 和 Kadota C：“妊娠中期感染鼠巨细胞病毒导致小鼠胚胎出现小眼症和脑萎缩。”

門田智佳,長浜真人,筒井祥博: "Relafionship between HOX2 homeobox gene expression and the human eytomegalovirus immediate enly genes" Journal of General Virology. 73. 975-981 (1992)

Tomoka Kadota、Masato Nagahama、Yoshihiro Tsutsui：“HOX2 同源盒基因表达与人类巨细胞病毒直接基因之间的关系”普通病毒学杂志 73. 975-981 (1992)。