Study On Site-specific Integration Of Salmon Calcitonin Or Human Serum Albumin Gene In The Goat Mediated By Cre/Loxp System

The random genome integration of transgene and its low efficiency are the main bottleneck problems to restrict the development of transgenic animal, but site-specific recombinase mediated cassette exchange reaction may be the way to realize the location of exogenous gene, and the LE/RE mutation strategy is expected to improve the efficiency of transgenic. Using the aboving two methods, the study develops a method of site-direct integration by Cre/loxp recombinase system.Firstly, the fetal fibroblasts of human lysozyme transgenic goat were separated.In these cells, the marker genes and loxp sites arrange in chains:5’-[loxplâ†'-Neo-TK-loxpâ†']-3’("â†'?"represents the orientation of the loxp sites), which is single-site integration. salmon calcitonin(sCT) located-integration vector (pTM-sCT2) with puroR gene was constructed, which contains the mutation loxp2and wild-type loxp site on both sides respectively. In the vector, sCT gene is in the downstream of bovine beta-lactoglobulin promoter.The study explored a way to accomplish site-direct integration by co-transfecting the Cre expression vector pBS185and pTM-sCT2into transgenic BLG3cells. The experiments explored6kinds of liposomal transfection conditions, among the results the best integration efficiency achieves40%.Another way to abtain the site-directed integration cell lines was explored by TAT-Cre. By electroporating,2positive clones of18transgenic integration cell clones were detected (the efficiency is11.1%), and by liposomal transfecting, the highest efficiency is20%. Comparing the aboving two methods, a way of site-direct integration was finalized: constructing the plasmid with target geneâ†'transfecting BLG3â†'incubating the transfected cell line by TAT-Cre for3h, piking clones by puromycinâ†'extracting DNAâ†'detecting the integration of target genes and the positioning integrationâ†'expanding culture and freezing the positive integrated clones.Secondly, in order to verify the influence of the different insert fragment size on integration efficiency, a vector called pTM-hSA2was constructed, in which the calcitonin gene (0.2kb) was substituted by the human serum albumin (4.0kb). Then by introducing TAT-Cre-mediated pTM-hSA2into BLG3, there are5positive ones of25abtained clones (the efficiency is20%).Finally, the positive clones was picked to somatic nuclear transplantation experim-entation. The results demonstrated that the fusion efficiency of cloned embryos and the develop efficiency of Mulberry blastocyst are consistent with that of normal somatic cell clone.In conclusion, the study has abstained two kinds of site-specific integration cell lines:sCT-located cell line and hSA-located cell line. And by Cre/loxp system, a method of producing located-integration transgenic animals was constructed, which can efficiently relise the integration of transgenes.