Studies On Animal Mammary Gland Tissue Specific Gene Expression At Targeted Loci

A 3.8kb bovine β-casein gene 5'regulatory region containing promoter, intron-1 and 5' untranslated region of bovine β-casein mRNA as well as known regulatory elements was obtained by PCR and DNA splice. Then the human serum albumin cDNA gene fused with a stretch of six consecutive histidine residues at its C terminus was introduced behind the bovine β-casein initiation codon ATG in frame. SV40 polyA signal and two loxP sites was added consecutively after the termination codon TAA, and then a GFP gene was inserted between the two loxP sites. After a lox2272 site was added in front of the bovine β-casein gene 5'regulatory fragment, a mammary gland universal expression vector pLbCAS-HSA-LGL was created.This expression vector pLbCAS-HSA-LGL has the following advantages: i) the 1.7kb promoter is able to drive cell-specific and hormone-dependent expression; ii) the inclusion of intron-1 can increase expression level of fusion genes; iii) the 5'UTR of bovine P-casein mRNA may have a positive role in both transcriptional and post-transcriptional regulation; iv) the GFP gene make the selection of positive clone among embryos possible; v) the GFP gene can be easily excised via Cre-mediated recombination between the two loxP sites after the expression vector has been integrated into chromosome; vi) the two incompatible lox sites, loxP and lox2272, would facilitate Cre-recombinase Mediated Cassette Exchange (RMCE), which in theory will leading to develop a technology of site-specific gene expression in animal mammary glands.