SAKAMAKI Kazuhiro Kyoto University, Graduated school of biostudies, Associate Professor, 生命科学研究科, 助教授 (20271017)
KONDO Sinae Kyoto University, Dep.Medicine, Associate Professor, 医学部, 助教授 (40314182)
GOTO Kazuo Central Institute of Experimental Animals, Dep.Genetics, Chief Scientist, 遺伝研究室, 主任研究員 (00205593)
|Budget Amount *help
¥16,800,000 (Direct Cost : ¥16,800,000)
Fiscal Year 2004 : ¥5,500,000 (Direct Cost : ¥5,500,000)
Fiscal Year 2003 : ¥11,300,000 (Direct Cost : ¥11,300,000)
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.