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

体内条件基因修饰技术，如条件敲除/过表达系统，具有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。我们的目的是简化用TAT-CRE融合蛋白进行体内基因修饰的复杂过程。首先，我们制作了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的反向版本）来评估体内基因重组。此外，我用CMV-Neo/pCX-LoxP-EGFP-LoxP-mRed电镀胚胎干细胞（ES）制备了嵌合体小鼠，并建立了小鼠系。通过将这些LoxP转基因小鼠系与普遍过表达的Cre转基因小鼠系（CMV-Cre）配对，我选择了一个系（CMV-Neo/pCX-LoxP-EGFP-LoxP-mRed）。另一方面，我们制作了三种不同的TAT-Cre融合蛋白（PDT1， PDT2和PDT3）。在体外检测中，由于PDT1和PDT2对水的稳定性较低，因此均制备了蛋白沉淀。为了提高PDT2在水中的稳定性，我们制备了聚乙二醇化的PDT3蛋白。随着聚乙二醇化PDT3蛋白水溶性的增加，PDT3蛋白的沉积速率很低。当小鼠同时注射PDT1和PDT2时，基因重组效率是分散的。腹腔注射聚乙二醇化的PDT3蛋白可提高基因重组效率。然而，聚乙二醇化的PDT3蛋白完全渗透到细胞中。本研究获得以下新知识，当将Cre融合蛋白注入妊娠小鼠体内时，该蛋白通过胎盘，胚胎基因组重组。我们希望在本研究期间实现无处不在的重组，但遗憾的是没有得出结论。

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.