Establishment Of Marker-free LacS Gene Transgenic Cow Cell Lines Using Specific Integrase

Transgenic animal is an important material in biological research, and selective marker gene is widely used in production process of transgenic animals.however, there remains biological safety risk in the presence of residual selective marker gene. Using Cre/Loxp recombinant system can achieve the purpose of selective marker gene deletion. In this study, pEGFP-N1-LacS vector is constructed, and co-transfected with phiC31 integrase mRNA in vitro transcription in cow fetal fibroblasts cells using electric transfection method. The LacS gene was integrated into the bovine fetal skin fibroblasts cells genome, then screened positive expression cell line stably express green fluorescence. Using Cre membrane penetrating peptide to made the green fluorescence were incubated, excision of selective marker gene, no green fluorescence in the cell lines stably transfected, for cultivate the marker free transgenic LacS gene cloned cows lay the foundation. The main results are summarized as follows:1. Placed the attB fragment into the pEGFP-N1 plasmid, and added two Loxps which are the same into the right sites of the plasmid. And the eukaryotic expression vector p-EGFP-N1-LacS was constructed.2. The pEGFP-N1-LacS plasmid was co-transfected into the cow fetal fibroblasts cells with phiC31 integrase mRNA using electric transfection method. Then screened the expression of green fluorescence cell line using 400μg/mL G418 culture.3. The pET-28a(+)-His-NLS-TAT-Cre plasmid was transformed into BL21 (DE3) competent cells. Then Cre protein was induced and purifited. Using the pure Cre transmembrane peptide to culture the cow cells witch express the green fluorescent protein. Then screened no green fluorescent cells were derived.