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The establishment of prion protein humanaized mice using homologous recombination

同源重组朊病毒蛋白人源化小鼠的建立

基本信息

批准号：
06404032
负责人：
KITAMOTO Tetsuyuki
金额：
$ 22.72万
依托单位：
Tohoku University (1996)Kyushu University (1994-1995)
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
1994
资助国家：
日本
起止时间：
1994 至 1996
项目状态：
已结题

项目摘要

To study human prions, we previously used the mice with wild type murine PrP gene for the transmission experiment. Mice have been used to do the transmission experiments from human or other animals with prion diseases. However, it takes long time for the successful transmission with the wild type mice, and the successful transmission was rarely observed in the wild type mice. Therefore, it is essential to establish humanized prion protein mice for the transmission experiment from human prion diseases. For this purpose, we designed the gene replacement method with the homologous recombination. Our method was based on the site-directed recombination using Cre-loxp system. At first, the loxp-neo-gpt-loxp cassette was used for the homologous recombination. One clone out of 140 G418-resistant clones was selected by the Southern blotting. We made the chimeta mice from this clone, and found out the germinal transmission from the chimera mice. The Cre plasmid was micro-injected in the fertiliz … More ed eggs from the heterozygous mice.In 6 out 7 mice, the successful site-specific recombination was observed. We established the humanized mice using the homologous recombination and the site-specific recombination. In addition, we used the loxp-neo-loxp cassette for making other types of humanized mice. With this cassette, the efficient homologous recombination was obtained. One out of 60 clones or 3 out of 120 clones were recognized positively by the Southern blot analysis. At present, we are making the chimera mice from these ES cells.For establishment of mice overexpressing human prion protein, we also made the transgenic mice. The natural promoter and the natural gene structure was obtained from the genomic library of the 129sv or I/ln mice. The transgene was constructed with the natural murine prion protein gene except for the exon 3. The exon 3 was replaced with human prion protein. Two constructs was micro-injected in the fertilized eggs from C57bl mice. We obtained 5 or 4 transgenic founder mice from each construct. These mice or germ-lined F1 mice showed a high expression of human prion protein mRNA.These replacement or transgenic mice were useful to analyze the human prion titer. Less

为了研究人朊病毒，我们先前使用了携带野生型鼠PrP基因的小鼠进行传播实验。小鼠已被用来进行人或其他动物朊病毒病的传播实验。然而，用野生型小鼠成功传播需要很长时间，并且在野生型小鼠中很少观察到成功传播。因此，建立人源化朊蛋白小鼠模型，为开展人朊病毒病的传播实验奠定了基础。为此，我们设计了同源重组的基因置换方法。我们的方法是基于使用Cre-loxp系统的定点重组。首先，将loxp-neo-gpt-loxp盒用于同源重组。通过Southern印迹从140个G418抗性克隆中选择一个克隆。我们用该克隆体制备了嵌合体小鼠，并从嵌合体小鼠中发现了生殖细胞的传递。将Cre质粒显微注射到受精卵中， ...更多信息艾德从杂合子小鼠的卵细胞中分离，7只小鼠中有6只成功地进行了位点特异性重组。我们利用同源重组和位点特异性重组建立了人源化小鼠。此外，我们使用loxp-neo-loxp盒来制备其他类型的人源化小鼠。利用该表达盒，获得了高效的同源重组。Southern印迹分析显示，60个克隆中有1个或120个克隆中有3个被阳性识别。目前，我们正在利用这些ES细胞制备嵌合体小鼠，为了建立高表达人朊蛋白的小鼠，我们还制备了转基因小鼠。从129 sv或I/ln小鼠的基因组文库中获得天然启动子和天然基因结构。用天然鼠朊病毒蛋白基因构建转基因，外显子3除外。外显子3被替换为人朊病毒蛋白。将两种构建体显微注射到来自C57 bl小鼠的受精卵中。我们从每个构建体获得5或4只转基因创始小鼠。这些小鼠或种系化F1小鼠显示人朊蛋白mRNA的高表达，这些替代或转基因小鼠可用于分析人朊蛋白滴度。少