Establishment Of Tissue-specific Thrombomodulin Overexpression Mouse Model

Objective1. To construct thrombomodulin (TM) expression plasmid and to analyze its biological activity in cells;2. To establish a tissue-specific TM overexpression in a transgenic mouse model and to analyze its biological activity in vivo.Methods1. Amplify TM gene fragment from lung tissue CDNA of wild type mouse;2. TA clone of TM gene into TOPO vector, transfect the corresponding plasmid into SVEC cells, then check cell proliferation, apoptosisand protein C activation assay;3. Subclone TM gene into PCCALL2 vector,co-transfect the corresponding PTMHL plasmid with Cre expression plasmid into SVEC cells, then test the TM expression andprotein C activiction;4. Make the transgenic mouse by pronuclear injection of PTMHL plasmid to determine the TM genotype of the mice by PCR, genomic DNA of mice was extracted from tails;5. Mate the new PTMHL transgenic mice with Tie2-Cre mice, determine the TM genotype and Cre genotype of the mice by PCR, genomic DNA of mice was extracted from tails, use Western blot and immunohistochemistry to detect TM expression in tissues, use protein C activation assay to detect the protein C activation in vivo.Results1. Successfully constructed the eukaryotic expression of TM plasmid, after transfected TM plasmid into SVEC cells, cell proliferation and apoptosis were inhibited, TM biological activity increased in the cells as well;2. Successfully constructed the tissue specific expression TM plasmid PTMHL, cotransfected PTMHL plasmid with Cre expression plasmid into SVEC cells, TM was only overexpressed in the presence of Cre, TM biological activity increased in the cells as well;3. Successful established a tissue-specific TM overexpression transgenic mouse model, after crossing with Tie-Cre mice TM expression on the endothelium of heart, kidney, liver, lung of the newborn mice significantly increased, the TM biological activity in increased in vivo as well.