Production Of Bovine Mammary Gland Bio-Reactor For Human Proinsulin Using Transgenic Cloning Strategy

Although pronuclear microinjection has been used for more than two decades to produce transgenic animal, the low efficiency and high cost of this technology has been the main barrier for transgenic animal production. The production of somatic cell clones derived from different tissue types of cultured cells opens new horizons for transgenic technologies. The advantage of using nuclear transfer to produce transgenic animals is the ability to use nuclei from preselected genetically modified donor cells. All of the animals created via NT from such selected cells should be but are not always transgenic. In the study present here, we constructed a transgenic vector for expression of human pro-insulin in mammary gland using GFP and neomycin resistant gene as marker genes. In vitro cultured bovine fetal fibroblast cells were transfected with this construct. The stable transfected cells were selected by G418 additton in the culture medium and GFP expression. Using the nuclei from selected donor cells, bovine blastocysts were produced via nuclear transfer technology and the blastocysts expressing GFP were transferred to recipients via embryo transfer technology. Experiment 1: Construction of transgenic vectorThe vector pO.8-IS containing 0.84kb of bovine a-lactalbamin promoter sequenceand 1.34kb of human pro-insulin genomic sequence (including exons, introns and 3'untranscribed region) in PUC18 backbone was excised as a 2.18kb of Hindlll-Kpnl fragment and inserted into the plasmid pGEM-7zf(+) that had been digested with Hindlll and KpnI. The vector was named p7Z-IS. The coding sequence of neomycin resistance gene was derived by PCR amplification from the plasmid pIRES2-EGFP and a Nhel site and a BamHI site was introduced respectly at the two ends of the sequence. Then the 942bp neomycin resistance gene fragment was assembled into the MCS of the plasmid pIRES2-EGFP that had been digested with BamHI and Nhel. The resulted vector which contains a Neor gene coding sequence and an EGFP coding sequence linked by IRES sequence in down stream of a CMV promotor was named pNIE. Then the vector pNIE was excised as an Nsil-Sspl fragment and inserted into the vector p7Z-IS which had been digested with Nsil and EcolCRI. So in the end we got the vector named pNEI, which contained the Neor gene and the EGFP gene regulated by CMV promoter for expression in a non-tissue specific mode and the human pro-insulin gene regulated by bovine a-lactalbamin promoter for expression specifically in mammary gland. The constructed vector pNEI, according to our design, was proved by restriction fragments using several endonuclear enzyme digestion and PCR amplification. Experiment 2: Isolation and in vitro culture of bovine fetal fibroblast cellThe bovine fetal fibroblast (BFFB) cells were isolated by attachment of tissue pieces from ear skin of a 3-4 months gestation bovine fetal. The cells grown into confluence 7 days after attachment and were cryopreserved after 2-3 passages forpurification and amplification. The growth curve of the cell was drawn during 7 days culture based on counting the cell number continually. The karyotype of the cell within 10th passages and after 20th passages was analyzed. As results the growth of the cell was very prosperous and more than 62% of the cells maintained diploid karyotype after 20th passages. So the bovine fetal fibroblast cells gave a good fit to transgenic cloning manipulation. Experiment 3: Gene transfection of the bovine fetal fibroblast cellFor transfection, the plasmid pNEI were linearized by digestion with Xhol and purified through phenol chloroform extraction. BFFB passage 3 cells in a 0100mm disk at 70% confluency were harvested and adjusted to 5x106cell/ml with HeBES. 200jlxI of the cell suspension were electroporated with 4 ug linearized pNEI vector in a 2-ram Gap cuvette for K 5> 1(K 15 and 20 ms at 800V/cm, 900V/cm, lOOOV/cm respectively. Cells were checked 24-48 hours after electroporation under fluorescence microscopy for GFP expression, and G418 selection (800ug/ml) was applied since then. After 2 weeks, selected colonies were counted and proliferated in culture medium containing 300 ng/ml G418 for 2-3 passages and cryopreserved. A small part of the cells were analyzed by PCR for gene integration. As results, bright green fluorescence can be detected 24 to 48 hours after electroporation, and more colonies were selected when electroporation parameters were 900V/cm, 5ms. The result of PCR detection showed that the foreign gene was integrated into the genome, and the selected cells should be competent for transgenic cloning. Experiment 4: Transgenic cell nuclear transferIn this study, we attempted to produce transgenic bovine mammary gland bio-reactor using somatic cell nuclear transfer and embryo implantation. Preimplantation screening by EGFP expression was used to detect NT-derived transgenic embryos to ensure that the newborn calf would be transgenic.Totally 3612 oocytes were aspirated from 2-8mm follicles of bovine ovaries collected from an abattoir, 72% of them were matured after 18 hours culture. We compared the parthenogenetic activation rates of 4 different treatments: 2uM Inomycin for 5 minutes, 7% ethanol for 7minutes, 2uM Inomycin for 5 minutes plus 2mM 6-DMAP for 4 hours and 7% ethanol for 7minutes plus 2mM 6-DMAP for 4 hours. There were no significant differences between the first 2 groups (25.7% vs. 33.7%) or between the last 2 groups (91.1% vs. 92.1%). However the parthenogenetic activation rates of the last 2 groups were significantly higher than those of the first 2 groups (P<0.01). We also compared the pronuclear morphology between the last 2 groups. The rate of 1 pronuclear formation of the Inomycin plus 6-DMAP group was lower than that of the ethanol plus 6-DMAP group. Totally 1443 oocytes were enucleated and 1119 enucleated oocytes were treated for electrofusion with donor cells. Among them, 654(58.4%) couplets were fused. Totally 594 reconstructed embryos were stimulated and cultured in vitro and 20% of them developed to blastocyst stage. We compared the fusion rates and the blastocyst formation rate between 2 different treatments of donor cells. One group of donor cells was normal cultured cells, and another group of donor cells was synchronized in Gi cycle stage by serum starvation for 2 days plus normal culture for 10 hours. There were nosignificant differences both in the fusion rate (55.3% vs. 56.2%) and in the blastocyst formation rate (23%vsl8.9%) between the two groups. Among the cloning blastocysts derived from transgenic cell nuclear transfer, 63% of them expressing GFP protein. OPS vitrification method was used for cryopreservation of the transgenic cloning blastocysts. The survival rate after thawing was 91%. 8 cryopreserved blastocysts were transferred into uterus of 2 recipient cows, but no pregnant was detected. Totally 21 freshly produced NT blastocysts expressing GFP were transferred into uterus of 7 recipient cows. 1 pregnant was detected after 3 months.