The Site-specific Integration Of Avastin And HLf By Cre/loxP System And RhLZ-AF

Cre/loxP mediated recombination reaction could be used to produce transgenic site-specific integration goats, this had been preliminarily validated in the human lysozyme transgenic goat fetal fibroblasts, and the sCT and hSA transgenic goats had been prepared. Fetal fibroblast cells were drawn difficulties, needed to be accurately identified gender and transgenic integration, and had other problems. This method needed to be further improved. Somatic cells were drawn easily, so it could make up for the deficiencies of fetal cells.In order to explore the feasibility of the transgenic site-specific integration goats preparation using adult fibroblasts, two mini-gene site-specific integration goats were studied in this research (human lactoferrin mini-gene and anti-vascular mini-gene endothelial growth factor monoclonal antibody); the gene designated method was explored in the somatic cells; the program for somatic cell cloning of transgenic site-specific integration somatic cells was optimized.The ear skin fibroblasts of recombinant human lysozyme transgenic goats 3-415 was isolated. The cells were identified that the genome contained the loxP sequence. The gene frame was 5’-[loxP1â†'-neo-TK-loxpâ†']-[human lysozyme expression frame]-3’. The ear skin fibroblasts of recombinant human lysozyme transgenic goats 3-415 had been isolated.A breast-specific expression vector (pTM-Asn) was constructed for Avastin. It’s frame was 5’-[loxP2â†'-polyA-Avastin-blg-Puro-loxPâ†']-3’. loxP1 was LE mutant, and loxP2 was RE mutant. Two ways of Cre was introduced to the ear skin fibroblasts for site-specific integration had been contrasted: â‘  The Cre eukaryotic expression vector pBS185 and pTM-Asn were co-transfected 3-415 cells with liposome transfection. The efficiency of site-specific integration was 23.08% (6/26). â‘¡The way of TAT-Cre protein transduction:200μg/mL TAT-Cre was added to mediate objective gene to realize site-specific integration after liposome transfection. The efficiency of site-specific integration was 13.64%(3/22). Monoclonal cell lines were screened and the efficiency of site-specific integration was detected. This showed that efficiency of co-transfected method of the pBS185 and site-specific integration vectors was higher.Another breast-specific expression vector(pTM-hLf) was constructed for hLF. Its frame was 5’-[loxP2â†'-polyA-hLf-blg-Puro-loxPâ†']-3’. Then pTM-hLf was transfected into 3-415 cells by the way of the pBS185 co-transfected. Cell clones were selected for 8-10 days by puromycin. Cells were expanded cultured until 6-well plates, then well growing cell clones were picked and detected by PCR/sequencing. The monoclonal cell lines that had Avastin and hLfgenes could be used for SCNT had been obtained by Cre/loxP system and hLZ-AF.The best hLf/Avastin positive clone cells was picked and used for nuclear transfer experiments. The rate of nuclear transplantation fusion for Avastin-hlz clone cells was detected. Reconstructed embryos were transplanted to the recipient goat’s womb.30 days after transplantation, The rate of pregnancy were detected by type-B ultrasonic. Detection result of an abortion showed that the target gene hLfwas integrated into the goats’ genome. The rate of nuclear transplantation fusion for Avastin-hlz clone cells reached 89.29%. The rate of pregnancy was 25.5%(14/55). The rate of nuclear transplantation fusion for hLf-hlz clone cells reached 77.72%(471/606). The Pregnancy rate was 20.83% (10/48). This indicates that the early pregnancy rate of the two genes targeting integration somatic cells cloned embryos were similar and within the efficiency range of somatic cells cloning in the pasture (14.4-42.9%). The target gene were detected by PCR in Avastin goat and hLf aborted fetus. Detection result of an abortion showed that Avastin and hLfwere integrated into the goats’ genome.In somatic cells, Cre/loxP also could highly efficient mediate site-specific integration of the transgene, different type of target gene was not obvious for reorganization, the obtained site-specific integration cells had no significant effect on somatic cell cloning efficiency. At the same time, anti-vascular endothelial growth factor monoclonal chimeric antibody (Avastin) cell clones/goats and human lactoferin cell clones/embryo had been obtained. It was provide a reference that establish a specific loci efficient expression transgenic livestock system.