Isolated bovine oviduct epithelial ceils by trypsin digestion from oviduct tissue of a 2-year-old dairy cow. Oviduct epithelial cells grow well in DMEM/F12 medium. The first generation oviduct epithelial cells were used as target cells to carry out electroporation transfection. Molecular size of the transfected genes is 31 085 bp. The gene is a plasmid that carry β-casein promoter,human collagen eDNA and EGFP,Neor as double marker. Electroporatioa experiment found that bovine oviduct epithelial cells can get positive-transfectant in hypotonic buffer. Among them,90 mOsm/kg is the best. For voltage,800 V is the best. High and low voltage are not conducive to the success of electroporation. Successfully transfected cells were screened with 800ug/ml of G418. on day 10, a larger cluster of positive cell clones were obtained. The cluster of positive clone cells were expanded to obtain a more pure cell line. Detected by flow cytomerty,the purity was 81.6% . This cell as a nuclear transfer donor carried out gene transfer experiments. The results showed that in transgenic cells and nontransgenic cells, their fusion rate of reconstructed embryos obtained significant difference（51.9 % vs 63.2 %）, their morula/blastocyst rate of reconstructed embrys were not significantly different （20.3 % vs 25.5% ）. DNA of the embryos displaying green fluorescence was analyzed,The results showed that the embryos was successfully transferred to the foreign gene and the genetic structure of the transfer was complete.

In this study,The COL1A1 cDNA gene was successfully cloned and ligated to the mammary gland specificity expression vector pGC1,Holstein cow fetal fibroblasts were transfected with the vector of pGC1-collagen by using electroporation,the transgenic cells were used as donor cells for nuclear transfer.After nuclear transfer the Holstein cow embryos carried the COL1A1 cDNA gene were developed and finally 8 reconstructed blastocyst were obtained in vitro.This demonstrate that the Holstein cow embryos originate from nuclear transfer and carried the COL1A1 cDNA gene can be developed normally.

In the article first PGC1-colleage was reconstructed with constructed expression vector and the humanⅠtype collagen gene,and then the ear fibroblast of Holstein cow was transfected in the liposome-mediated method,optimized a series of transfection conditions to enhance the transfection efficiency,the stable monoclone cell line expressed the green fluorescent protein which was observed with G418 and fluorescence microscope,the normal untransfected fibroblast was the control,before transfection the authenticated archae-plasmid was the positive control,then amplified and cultivated,at last extracted the genome DNA and PCR authentication.The result indicated that the cell degrees of fusion was about 50%～60%,mediated 1~3 μg recombinant plasmid,transfeced 24 h,transfection efficiency was 25%～30% in the 6-hole culture plates,monoclone cell was screened by that detected the expression of the green fluorescent protein with fluorescence microscope.In our laboratory before the experiment,did the cell G418 tolerance experiment,indicated that 800 U G418 caused drug-resistance and screening effect was the best,maintained concentration 300 U～600 U.25 green fluorescent protein positive clone was selected from the ear fibroblast of Holstein cow,selected 6 clone to do PCR experiment: authenticated the screened clone,extracted the genome DNA,PCR result accord with the expectant band,indicated that humanⅠtype collagen gene was transfected into the ear fibroblast of Holstein cow in the liposome-mediated method,screened the positive clone that expressed the gene stably.PCR authentication established some foundation for the transgene clone animal.

The objective of this study was to develop an effective method to screen transgenic donor cells and improve the production efficiency of transgenic embryos. In the study, goat (Capra ibex) skin fibroblasts were co-transfected with two expression constructs, one containing the human α1 (I) procollagen cDNA fragment (4.3kb) and the neomycin resistance (Neor) gene, and the second containing the green fluorescent protein (GFP) gene. The Neor and GFP genes were used as markers for the screening of donor cells. Results revealed a significant difference (P<0.01) in the production rate of positive transgenic blastocysts (44.6% versus 82.8%) between donor fibroblasts selected using antibiotic G418 alone (Treatment A) and those from donor fibroblasts selected using antibiotic G418 and GFP (Treatment B), respectively. No significant differences were found between Treatments A and B in the total somatic cell nuclear transfer (SCNT) embryo cleavage rates (71.1% versus 65.9%) and total SCNT blastocyst formation rates (18.2% versus 15.7%). The research results indicate the Neor and GFP genes located at different constructs can be effectively combined by co-transfection to screen transgenic donor fibroblasts, and increase the yield of transgenic SCNT embryos carrying human α1 (I) procollagen cDNA in goats.

Through isolation and pure culture of Holstein milk cow ear fibroblasts and analyses research on several cryopreservation methods in this studing, the results show the satisfactory primary culture of Holstein milk cow ear fibroblasts had been obtained by small tissue cubes culture with complete medium (DMEM/F12+20%FCS+100IU/mL(P/S). Most fibroblasts could be collected in the digestion of primary fibroblast mass cultures by TrypsinEDTA (0.25% Trypsin,1mM EDTA.4Na) , through 2 to 3 subcultures the pure culture of Holstein milk cow ear fibroblasts could be implemented .On the conditions of the same cryoprotectant and FCS concentration Holstein milk cow ear fibroblasts were cryopreservated in five cryopreservation methods, through the contrast and analyses of cell vigor after five months cryopreservation , method 3 (70% cell suspension +20%FBS+10% DMSO ; cryopreservation procedure: First fibroblasts were pre-cooled and balanced for half hour at 4 ℃, and second hanged in gaseous nitrogen in close to the mouth of nitrogen canister for 4 hours ,and finally sunk into liquid nitrogen.) was the best cryopreservation method , it could maintain 84.8% of living rate after cells were revived and stained by Trypan Blue and 85.4% of adherence rate after cells were revived and cultured for 48 hours.

In this study using the transgenic(GFP) fibroblasts of Saanen dairy goat as donor cells and using the abattoir-derived oocytes of normal goat as recipient cells the nuclear transfer experiments had been processed.During experiments the optimization of electricfusion experiment(parameters) of transgenic nuclear transfer embryos had been done.For the treatment of transgenic reconstruction embryos of Saanen dairy goat,the electricfusion parameter of group B in(experiment) plans(1.5kV/cm for 30 μs and pulse interval for 1s) is most suitable for the(transgenic) reconstruction embryos of Saanen dairy goat,the electricfusion rate of 89.4% for cell-cytoplast complexes and the cleavage rate of 68.0% for reconstructed embryos have been obtained.

以新疆野生盘羊（Ovis ammon）和萨能奶山羊转基因（GFP）成纤维细胞为核供体细胞,分别以绵羊卵母细胞和普通山羊卵母细胞为受体细胞进行了核移植,对不同种类核移植重构卵进行电融合处理,发现盘羊重构卵电融合参数为1.4 kV/cm、20μs/次、间隔1 s时的重构卵融合率最高;对于转基因（GFP）萨能奶山羊重构卵的处理,电融合前先放入融合液中于38.5℃下平衡5 min,融合时电融合参数为1.5 kV/cm、20μs/次、间隔1 s时的融合率最高。实验中发现将融合后的两种重构胚胎先置入IVM培养液中于38.5℃下平衡4 h,可降低重构胚胎的死亡率,提高发育质量和卵裂率。

In this study the synchronized estrus of sheeps were induced by CIDR+PMSG,experiments of ovulation induction were performed on 86 of Recipient Sheeps by difference LH dosages with three methods(Method 1 100IU,Method 2 200IU and Method 3 300IU).The results revealed that the percentages of synchronized estrus were 95.2% totally,percentage of synchronized estrus at the first day was 61%,percentage of synchronized estrus at the second day was 39%,percentage of synchronized estrus at the first day was obviously higher than at second day(P0.01).High quality corpus luteum(55.2%) which ovarys contain through the treatment of method 1 was significantly superior to that(26.9%) through the treatmentof method 2(P0.05),but no striking diference existed between method 1 and method 3,in experiment more chemical could be saved by using method 1.

Through isolation and pure culture of Shaneng milk goat fibroblasts with skin tissue, fibroblast line was established. On conditions of the same cryoprotectant and FCS concentration Pure Shaneng milk goat fibroblasts cultured were cryopreservated in five cryopreservation methods with different pre-cool and balance ways and pre-cool time, through the contrast and analyses of cell vigor after five months cryopreservation, method 3 (70% cell suspension + 20%FCS + 10% DMSO; cryopreservation procedure: first fibroblasts were pre-cooled and balanced for half hour at 4 C , and second hanged in gaseous nitrogen in close to the mouth of nitrogen canister for 4 hours, and finally sunk into liquid nitrogen. ) was the best cryopreservation method, it could maintain 84. 8% of living cell rate through cell vigor test after cells were revived and stained by Trypan Blue and 85. 4% of cell adherence rate after cells were revived and cultured for 48 hours, living cell rate and cell adherence rate of method 3 superior to other four methods.

为了对在春季屠宰场所采绵羊卵母细胞的分布及不同分布的成熟率有明确的认识,2003年3月至4月共进行15次试验,收集卵母细胞1 485枚.按卵母细胞外层所含卵丘细胞及完整程度分成A,B,C,D,E 5个级别.A级:卵丘细胞层完整且紧密,一般为4层以上;B级:卵丘细胞层完整,一般为1～3层;C级:有卵丘细胞包被,但不完全;D级:裸卵,但细胞质均匀,细胞形态完好;E级:退化裸卵,胞质不均匀,细胞形态消失.选择A,B,C,D级卵母细胞单独进行体外成熟培养,培养液为TCM-199十5%FCS+5μg/mL LH+5 μg/mL FSH+1 μg/mL β-estradiol+2%penstrep,培养条件为5%CO2,38.5℃,饱和湿度.结果显示;A,B,C,D级的分布分别为19.8%、28.1%、19.6%和32.4%,成熟率分别为81.3%、65.3%、61.5%和17.0%.总体成熟率为52.1%,卵丘-卵母细胞复合体(COCs)的成熟率为68.9%.