Studies on In Vivo gene modification by injection of TAT-Cre fusion protein and establishment of the practical technique.

在体内，条件性基因修饰技术，如条件性基因敲除/过表达系统，有一个Cre/loxP系统。在大多数情况下，小鼠的Cre和loxP系是独立发育的，然后杂交。我们的目的是简化TAT-Cre融合蛋白体内基因修饰的复杂过程。首先，我们建立了6个不同的转基因小鼠系(PCX-loxP-EGFP-loxP-dsRed2，PCX-loxP-pd4EGFP-loxP-dsRed2，PCx-loxP-EGFP-loxP-mred以及GFP和RFP各自的反向版本)。在活体基因重组中进行了评估。在此基础上，将CMV-Neo/PCX-loxP-EGFP-loxP-mRed电镀胚胎干细胞构建成嵌合体小鼠，建立了小鼠系。通过将这些loxP转基因小鼠品系与一个普遍过表达的Cre转基因小鼠品系(CMV-CRE)交配，我选择了一个品系(CMV-Neo/PCX-loxP-EGFP-loxP-mRed)。另一方面，我们制备了三种不同的TAT-Cre融合蛋白(PDT1、PDT2和PDT3)。在体外实验中，由于PDT1和PDT2对水的稳定性较低，所以都进行了PDT1和PDT2蛋白的沉淀。为了提高PDT2的水稳定性，我们制备了聚乙二醇化的PDT3蛋白。随着聚乙二醇化的PDT3蛋白的水溶性增加，PDT3蛋白的沉淀率很低。当PDT1和PDT2同时注射到小鼠体内时，基因重组效率分散。小鼠腹腔注射PEG化PDT3蛋白后，基因重组效率提高。然而，PEG化的PDT3蛋白完全渗透到细胞中。本研究获得了以下新知识：当Cre融合蛋白注射到怀孕小鼠体内时，该蛋白正在穿过胎盘，重组胚胎基因组。我们希望在这段时间内实现无处不在的重组，但遗憾的是没有提供结论。

In Vivo conditional gene modification technology, such as conditional knockout/overexpression systems, has a Cre/LoxP system. In most cases, Cre and loxP lines of mice are developed independently, and then crossed. Our purpose is to simplify a complicated process of an In Vivo gene modification with TAT-CRE fusion protein.The first, we made different six transgenic mice line (pCX-LoxP-EGFP-LoxP-dsRed2, pCX-LoxP-pd4EGFP-LoxP-dsRed2, pCX-LoxP-EGFP-LoxP-mRed and a reverse version of each of GFP and RFP, respectively.) which evaluated In Vivo gene recombination. Furthermore, I made a chimera mouse from the CMV-Neo/pCX-LoxP-EGFP-LoxP-mRed electroplated embryonic stem cell (ES) and established a mouse line. By mating of these LoxP transgenic mice lines and a ubiquitously overexpressed Cre transgenic mouse line (CMV-Cre), I selected a line (CMV-Neo/pCX-LoxP-EGFP-LoxP-mRed). On the other hand, we made three kinds of different TAT-Cre fusion proteins (PDT1, PDT2 and PDT3). In Vitro examination, both PDT1 and PDT2 protein sediment was made, because the stability for water was low. To increase the PDT2 water stability, we made PEGylated PDT3 protein. As water solubility of PEGylated PDT3 protein was increased, the sediment rate of PDT3 protein was very low. When both PDT1 and PDT2 were injected to the mice, gene recombination efficiency was scattered. Intraperitoneal injection of mice with PEGylated PDT3 protein results in the gene recombination efficiency was increased. However, PEGylated PDT3 protein permeates into cells completely.The following new knowledge was acquired in this research, when the Cre fusion protein was administrated into pregnant mice, the protein was passing through the placenta, recombination of an embryo genome. We would like to realize the ubiquitously recombination within this research period, but unfortunately a conclusion is not provided.