| The end of this experiment is using Nuclear Transfer and recloned to product in vitro goat embryos of transfer t-PA foreign gene, and make transgenic animal. The study is to examine foreign gene integration and expression in preimplantation embryos in goat by fluorescent and PCR examining. It is to enhance blastocyst rate of nuclear transfer embryos by making in vitro culture system better. (1) Goat oocytes were cultured in maturing media(OM) adding equal concentration (10 ng/mL) (EGF), (IGF),or (EGF+IGF). The matured rate(80.0%, 82.4%) of goat oocytes in OM adding IGF and (EGF+IGF )were higher than that in OM adding EGF(73.8%)(P>0.05), and higher significantly than that in OM(67.3%) (P<0.05). (2)Season can influence the matured rate of goat oocytes. The study show, from December to next year January, the matured rate of goat oocytes was the highest (81.0%); the matured rate of goat oocytes was 74.1% in November; it is the lowest (55.1%) from June to July. (3)pH of OM media can influence the matured rate of goat oocytes. When pH of OM is 6.8～7.0 and 7.2 , the matured rate of goat oocytes were higher significantly than that is alkaline (≥7.5). 2. Oocytes of goat were activated by the way of using Eth + 6-DMAP ,A23187+6-DMAP and Ion+6-DMAP. The cleavage rate and the blastocyst rate of oocytes activated by Eth + 6-DMAP (35.2%,4.8%)were significantly lower than the cleavage rate and the blastocyst rate of oocytes activated by A23187+6-DMAP(68.9%, 24.1%) and Ion+6-DMAP(88.1%, 48.0%). When embryos were co-cultured with GCs in media CR1aa and media SOFaa, the same concentration (10%) OCS, FBS and NCS were added . When embryos co-cultured with GCs in media CR1aa, the blastocyst rate of parthenogenetic embryo added OCS and FBS (48.1%,45.0%) were significantly higher than the blastocyst rate of parthenogenetic embryo added NCS(26.0%). When embryos co-cultured with GCs in media SOFaa, the blastocyst rate of parthenogenetic embryos added OCS and FBS (50.5%,48.7%) were significantly higher than the blastocyst rate of parthenogenetic embryos added NCS(29.30%). It was concluded that, both CR1aa and SOFaa could be used as goat parthenogenetic embryos in vitro culture; the blastocyst rate of parthenogenetic embryos were significantly increased when added 10% OCS and 10%FBS, nevertheless , 10% OCS was better. 3. the cow β-lactoglobulin gene 5′flanking fragment which was cloned by other researcher in our laboratory was reformed and was cloned at T site of pMD 18-T Vector. The positive recombination plasmid was marker as pBLG. pBLG and pEGFP-c1 were digested with restrictive enzyme Xhol I and EcoR I, the aim fragment was connected with T4 DNA ligase, the positive recombination plasmid was marker as pEB. pEB and ptPA were digested with SalI and BamH I, the aim fragment was connected with T4 DNA ligase, so the expressional vector was constructed and was marker as pBT 4. The nuclear transfer of transgenic somatic cell is an effective method for making transgenic animal with efficient expression of foreign gene. Transgenic structure of t-PA was built which contained positive marker genes(noer and GFP).The Method of lipofectamin were used to transfect the goat fetal fibroblasts , positive cell were see fluorescent protein(GFP) for 48 h. G418 was used to sieving and clone cell for 33 days, foreign gene was integrated to cell by PCR examining. Positive fetal fibroblasts were frozen , the live rate of thawed positive fetal fibroblasts is 48%. Cells can passage 5 passages. 5. The effect of location of polar body , time of oocytes maturation and CB on Denucleaus of oocytes were studied. Denucleaus rate was 89.7% when location of polar body was located in 4～5 site of clock which was significantly higher than when location of polar body was located in 3 site of clock (79.6%). The result proved that the method of Denucleaus used in this research was effectively. CB and Sucrose+CB groups Denucleaus rate(82.4%, 89.7%)was significantly higher than control group (67.3%);Cleavage rate (80.4%, 82.4%)and blastocyst rate (18.1%, 19.5%)of the cloned embryos were significantly higher than control group(73.3% and 9.4%)。Denucleaus rate (90.3%) was highest when the time of oocytes maturation was 18 h, which higher than 20-22 h(86.1%),which was significantly higher than 24 h. Blastocyst rate of the cloned embryos matured for 22~24 h was 19.6%, that is higher than these matured for 18 h (17.5%)and 24 h(15.9%). 6. (1)The development of nuclear transfer embryos derived from fetal fibroblasts including t-PA gene, fetal fibroblasts and ovary granulose cells were compared. The cleavage rate (82.9%)and Morulae rate (55.9%)of ovary granulose cells were higher than that of fetal fibroblasts (75.5%,50.0%) and fetal fibroblasts including t-PA gene (75.7%,48.7%); Blastocyst rate (19.7%) of ovary granulose cells was higher than that of fetal fibroblasts (16.6%) (P>0.05), and significantly higher than that of fetal fibroblasts including t-PA gene(11.0%) (P<0.05). (2)The development of nuclear transfer embryos derived from fetal fibroblasts including t-PA gene in media DMEM/F12 including 0.5%FBS for starvation and including 10%FBS were compared. The cleavage rate(75.6%) of nuclear transfer embryos in media DMEM/F12 adding 0.5%FBS is not significantly different from that (75.8%)in mediaDMEM/F12 adding10%FBS; Morular rate (46.5%) and blastocyst rate (11.0%) in media DMEM/F12 including 0.5%FBS were higher than that (39.3%, 9.0%) in media DMEM/F12 adding 10%FBS (P>0.05).Using transgenic fetal fibroblasts to make nuclear transfer embryos, witch was see different measure fluorescent protein (GFP). It shows that the nuclear transfer of transgenic somatic cell is an effective method for making transgenic animal. 7. The in vitro development of goat embryos, that obtained by nuclear transfer of transgenic somatic cell, were investigated in present experiment. The results showed that the blastocyst rate was significantly higher when the embryos co-cultured with granular cells (GCs) in CR1aa supplement with 10% bovine follicular fluid (BFF) (10.8%) than that in 5% BFF (7.8%) and 15% BFF (2.8 %). When embryos co-cultured with GCs in media SOFaa, the highest blastocyst rate was obtained from 10% BFF supplement (11.2%), which was significantly different from 5% BFF group (8.8%) and 15% BFF group (3.0%). Adding 10% BFF and 10% FBS into media CR1aa and SOFaa respectively, the goat blastocyst rates were 10.5%,11.0% and 5.5 %,5.8%. It was concluded that, both CR1aa and SOFaa could be used as goat transgenic cloned embryos in vitro culture; 10% BFF could significantly increase the blastocyst rate; BFF was more efficient than FBS for the early development in vitro of goat transgenic cloned embryos. 8. (1) Goat cloned embryos were compared with recloned embryos in ability of in vitro development. Fetal fibroblasts including t-PA gene was microinjected into cytoplasm of the MII oocytes. Goat embryos (G0) were cloned by this procedure. A single blastomere from 8～64-cell goat cloned embryos(G0) was microinjected into Intracytoplasm of the MII oocytes. Goat embryos (G1) were cloned by this procedure. Goat embryos (G2, G3 G4) were recloned by using 8～64-cell recloned embryos . The cleavage rate of cloned embryos(G0) (76.4%)was no difference significantly with recloned embryos (G1 G2 G3G4) (72.0%,76.0%,75.9%,74.8%). The developmental rate of morulae and blastocysts of cloned embryo(s47.1%,11.0%) were higher than these of recloned embryos(34.9%,28.2%,23.3%,22.5%)(3.9%,2.0%,0,0). (2) The developmental time of donor affected The developmental rate of recloned embryos(G1,G2). The morulae,blastocysts rate(29.6%,24.3%),(2.0%,1.9%) of recloned embryos (G1 G2) from 8～16-cell recloned embryos was lower than that(34.3%,29.9%),(3.9%,3.4%)from 32～64-cell recloned embryos(P>0.05).
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