Human Gdnf Gene Knock-in At Beta-casein Locus In Bovine Fetal Fibroblast Cells And Production Of Gene-Targeted Blastocysts By SCNT

The highlight has been given to the recombinant human proteins produced by the mammary glands of genetic,ally modified transgenic livestock mammals in the top field of biological technology.However,for random integration of mammary gland bioreactor,inserted gene expression level varied and stayed at a low level because of affecting by flanking DNA sequences.In contrast with random integration,site-specific integration of mammary gland bioreactor created by gene targeting at high expressing locus can remarkably improve gene expression.Glial cell line-derived neurotrophic factor(GDNF),a distant member of the transforming growth factor[beta](TGF-[beta]) super family,is a novel type of neurotrophic factor cloned in 1993.Researches revealed that it may have potential application in the treatment of Parkinson's disease and other diseases of the central nervous system as a survival factor for central dopaminergic neurons.In the present study,we constructed a gene targeting vector for the human gdnf gene knock-in at the bovine beta-casein gene locus so that human GDNF protein can be produced at high level in the mammary gland of the gene-targeted bovine.Human mammary tumor epithelial cell line Bcap-37 cells were used to analyze the rationality and validity as well as the biological function of the vector.Then bovine fetal fibroblast cells were transfected with the plasmid DNA.The gene-targeted fibroblast cells were obtained after positive-negative selection and PCR identification.The gene-targeted cells were used as nuclear donor to produce gene-targeted blastocysts by nuclear transfer.The results lay a massive foundation for production of human GDNF protein by gene-targeted bovine mammary gland bioreactor.1.Construction of targeting vector pNRTCNbG for the human gdnf gene knock-in at the bovine beta-casein locusThe pPGK-neoLoxP vector containing neo gene between two LoxP sites was used as plasmid backbone to construct the targeting vector pNRTCNbG.The 5' and 3' homologous arms,the beta-casein gene fragments,were amplified from purified bovine genomic DNA by PCR.The 5' homologous arm was 2.2 kb fragment including promoter,exon 1,intron 1 and part of exon 2 of the bovine beta-casein gene sequence,and the 3' homologous arm was 5.7 kb fragment including the bovine beta-casein gene 3' flanking sequence.The human gdnf cDNA amplified by RT-PCR was located at the downstream of the 5' arm.Moreover,SV40 polyA signal sequence was located at the downstream of the human gdnf gene as its transcriptional ending signal.The neo gene,positive selection marker gene,was located between the 5' and 3' homologous arms.The HSV-tk gene and DsRed2 gene were located outside the homologous recombinant area as negative selection marker genes,respectively The recombinant plasmids were identified by restriction fragment analysis and partial DNA sequencing.The results show that the structure of the final constructed vector accords with the designed plasmid map.2.Analysis of the biological function of the gene targeting vector pNRTCNbGIn order to analysis the bioactivity of the vector,linearized pNRTCNbG was transfected into human mammary tumor epithelial cell line Bcap-37 by using lipofectamine.After selection with G418 for 8-10 days,cell clones expressing red fluorescence protein were obtained.PCR analysis demonstrated that gdnf cassette had integrated into the genomic DNA of the transfected Bcap-37 cells.After proliferation culture,the transgenic cells were cultured in induction medium containing serum-free RPMI-1640 medium with prolactin,insulin and hydrocortisone,which can induce recombinant human GDNF expression.RT-PCR and Western-blotting analysis showed that the induced cells produced recombinant human GDNF mRNA and protein.The results show that the constructed targeting vector pNRTCNbG has bioactivity to efficiently express GDNF in mammary gland cells and secrete the protein outsite of the cells.At the same time,it is first time to confirm that human mammary tumor epithelial cell line Bcap-37 is valid for bioactivity analysis of mammary gland specific expression vector.3.Human gdnf gene knock-in at beta-casein locus in bovine fetal fibroblast cells The fetal fibroblast cells were successfully isolated from bovine fetus tissue.The gender of fetuses was identified using PCR.The fibroblast cells from the female fetus were cultured consecutively for 75 days.Morphological observation and analysis of chromosome number were carried out.The results showed that the cells possessed normal morphology,proliferation characteristics and chromosome number.So the bovine fetal fibroblast cells gave a good fit to gene targeting manipulation.DsRed2 was used as a marker gene for negative selection in the present study for the first time.In order to compare enrichment efficiency of targeting events between selections with DsRed2 gene and HSV-tk gene,linearized pNRTCNbG was introduced into the bovine fetal fibroblast cells by electroporation.The transfected cells were divided into two aliquots.The cells in each aliquot were selected by addition with G418 or G418 plus GANC in cell culture medium,respectively.After selection with G418 or G418 plus GANC for 8-10 days,the resistant clones in which targeting events had occurred were enriched by eliminating cells clones expressing red fluorescence protein via a fluorescent microscope.The results indicated that enrichment factors for DsRed2 and HSV-tk used as negative selection marker genes were 4-fold and 2-fold, respectively.Moreover,compared with GANC,DsRed2 protein had not cytotoxic effect on those gene-targeted clones.Subsequently,3.2×10⁷ bovine fetal fibroblast cells were transfected with linearized pNRTCNbG by electroporation.After selection with G418 for 8-10 days,total 3057 cell clones were obtained.After eliminating the cell clones expressed red fluorescent protein under a fluorescent microscope,773 cell clones were harvested,in which 5 cell clones involved in gene targeting events were conformed by PCR analysis and PCR product sequencing.Therefore,the relative targeting frequency was 0.65%(5/773),and the absolute targeting frequency was 1.6×10^-7(5/3.2×10⁷)4.Preparation of human gdnf gene-targeted blastocysts by somatic cell nuclear transferThe cloned blastocysts were prepared by somatic cell nuclear transfer,using the gene-targeted cells as the nuclear donor cells and enucleated oocytes matured in vitro as the recipient cytoplasm.In order to make sure the proper method of activation and development culture of reconstructed embryos,activation and development culture of parthenogenetic oocytes were carried out.The oocytes matured in vitro were activated with 7%ethanol for 7 minutes followed by incubation in SOFaa-BSA medium containing 2 mM 6-DMAP for 4 hours.After the activation the embryos were cultured in SOFaa-BSA medium for 36 hours,the cleaved embryos(cleaved rate was 74.9%) were co-cultured with cumulus cells in SOFaa medium containing 4%FBS for 7 to 9 days.The blastocyst formation rate was 24.9%.The results indicated the procedure could be used for activation and in vitro development of reconstructed embryos.In the present study,total of 2627 bovine oocytes were aspirated from follicles(2-8 mm in diameter).70.2%of them were matured after 18 hours culture.186 in vitro matured oocytes were enucleated, and 154 of them were electrofused with donor cells.Among them,134(87.0%) couplets were fused.134 reconstructed embryos were activated and cultured in vitro,and 5(3.7%) of them developed to blastocyst stage.2 of the blastocysts were chosen at random to detect whether the cloned blastocysts were transgenic blastocysts by PCR.The results showed that all of them were cloned transgenic blastocysts.