Ht-PAm Gene Knockin Goat Beta-casein Locus And Nuclear Transfer Of Gene-targeted Cells

The production of recombinant protein is one of the major successes of biotechnology; animalcells are required to synthesize proteins with the appropriate post-translational modifications.Transgenic animal mammary gland bioreactors are being used for this purpose. Producingmammary gland bioreactor showed great advantage over many years, but the level of transgenicexpression was low in transgenic animals and the diversity was greater because of the positioneffect of transgene and the artificial recombination of the gene elements. Gene targeting based onthe principle of gene homologous recombination had been studied and applied, because thetransgene could be integrated precisely in the chromosome. Gene targeting is a more powerfulmethod to produce mammary gland bioreactor, and nuclear transfer from cultured somatic cellsprovides an wonderful means of cell-mediated transgensis. Here we describe efficient andreproducible gene targeting in goat fetal fibroblasts to place the human tissue-type plasminogenactivator mutant (ht-PAm) cDNA at the beta-casein locus, and would produce the transgenic goatby nuclear transfer. To construct the gene targeting vector pGBC4htPAm, the Guanzhong milk goat beta-caseingenomic DNA sequence for homologous arms had been cloned firstly. The left arm was 6.3 kbfragment including goat beta-casein gene 5?flanking sequence, and the right arm was 2.4 kbfragment including beta-casein gene from exon 8 to exon 9. The ht-PAm cDNA was subcloned inthe goat beta-casein gene exon 2. The bacterial neomycin (neo) gene as positive selection markergene, was placed in the beta-casein gene intron 7, the thymidine kinase (tk) as the negativeselection marker gene, was just outside the right arm. The validity of the positive-negativeselection vector was tested , and targeting homologous recombination were elevated to 3.7-foldwith the tk gene. The DNA fragment between two loxP sequences was deleted effectively usingCre recombinase in vitro. Goat fetal fibroblasts were isolated from day 35 fetuses of Guanzhong milk goats andcultured to subconfluence before transfection, about fibroblasts were electoporated with linearpGBC4htPAm. Transfected cells were cultured in 96-wellplate for 24 h without selection, thenadded the drug G418(600 μg/ml) and GANC(2 μmol/l). With LipefectaminTM-2000, cells weretransfected with linear pGBC4htPAm. These transfected cells were cultured for 24 h withoutselection, then added the drug G418(800 μg/ml). After 12 days of selection, well separated cloneswere isolated and expanded in 24-wellplate, then added the drug G418(300 μg/ml) and GANC(2μmol/l). 1204 clones were selected, and only 656 clones could grow and be tested by PCRscreening for targeting. The results demonstrated that 48 targeting cell clones with homologousrecombination events were obtained using 3?-PCR, and 3 cell clones were verified by DNAIV ht-PAm 在山羊β-casein 基因座定位整合与中靶体细胞核移植的研究sequence analysis on the homologous recombination region, 2 ones were verified by 5?-PCR. Goat cumulus-oocyte complexes (COCs) recovered by surface-follicles cutting frombreeding seasons were matured in vitro, and 71.2% COCs were matured. The 3 targeted cellclones were nuclear transfer as donor cell after 1~3 days culturing with 0.5% FCS, and thereceptor cells were MII oocytes without nuclear and first polar bodies. These reconstructedembryos were fused for 40 μs under 1.2 kv/cm voltage, and activated using 5 μmol/l Ionomycinfor 5 min,2 mmol/l 6-DMAP for 4 h. 55.2% reconstructed embryos were fused and co-culturedwith cumulus cells in CR1aa. The results showed that the percentages of fused embryos weredifferent from different oocyte types (matured in vivo or in vitro) and different transgenic somaticcell lines. 600 reconstructed embryos had been obtained, and some could develop to morula orblastocyst in vitro. These developed cloned embryos were transferred to 16 recipients, and 6recipients did not returned...