| Since the birth of Dolly in1997, somatic cell nuclear transfer (SCNT) technique has successfully produced many animal species. However, the low efficient production of healthy cloning animals has made this technology extremely limitation, especially when combined with transgenic technique in farm animals. A series of problems such as low pregnancy, high abortion, abnormal fetal development, high birth-death, and also the low expression or silence of transgenic foreign genes emerged, which made it much more difficult in the application of SCNT and/or transgenic techniques. In this study, we systematically modified almost every steps of SCNT in bovine and established a efficiency increased bovine SCNT system. Using this modified protocols a large number of cloned embryos were obtained and transgenic cloning offspring were produced.Total of33043oocytes were micro-manipulated and26548fused embryos were obtained and cultured. We got7519of all kinds of transgenic cloned blastocysts. A number of1523recipient cows were transplanted with transgenic cloned embryos, among which414were pregnant, the pregnancy rate was27.2%.1. Modifications of bovine somatic cell nuclear transfer protocols Several modifications of bovine SCNT protocols were successfully conducted. Two-step oocyte maturation in vitro, optimized parthenogenetical activation time-point, and twice ionomycin stimulation of cloned fused embryos had made increased embryo development, improved embryo quality and higher pregnancy after embryo transfer. The results showed that1) compared with the one-step maturation protocol, the cumulus cells were denuded after maturation of the oocytes for20-22h, the two-step maturation procedure, that is, the oocytes were denuded when the oocytes were cultured for16h, then the denuded oocytes were continued to culture up to20-22h. The enucleation of the two-step matured oocytes was95.1%, which significantly higher than the one-step matured oocytes (74.3%). The blastocyst development was also higher in two-step oocytes (46.9%) than the one-step oocytes (36.5%, P<0.05).2) Interval between fusion and activation affected the embryo chromatin/chromosome configuration. Immunohistochemical analysis showed that the premature chromosome condensation (PCC) were96%and80%, respectively, in the reconstructed embryos after fusion for1h and2h. However, when the fused embryos were further cultured3h or4h the PCC occurrences decreased lower than50%, scattering or multi-elongated chromosomes were observed. When the interval time prolongs to5h to6h, more than80%of the embryos appeared chromosomes scattering or dividing into several pieces. The interval between fusion and activation less than2h was the best choice before activation3) Activation of the fused embryos at22,24,25,26,27,28h after initiation of maturation resulted in different embryo development, and activation at26h after oocyte maturation obtained the highest blastocyst development.4) Twice treatment of fused embryos with ionomycin significantly increased embryo development and establishment of transferred embryos pregnancy.2. The match of cloned embryo status to recipient cow estrusThe match of developing embryos to recipient cow estrus status would improve successful establishment of pregnancy. We chose embryos at morula, early blastocyst, blastocyst,expanded blastocyst and hatching/hatched blastocyst to transfer into uterus of recipient at different estrus stages. The results showed that1) The highest hatched blastocyst rate were obtained of the blastocysts emerged on Day6of culture had when compared to those blastocysts occurred on Day7,8and9d (82.4%vs.46.2%vs.16.1%vs.7.1%, P<0.01). Day6blastocysts resulted in significantly higher pregnancy than the other groups.2) In order to examine the best matching between embryos and recipient cow,the embryos at different developmental stage were transfered into recipients that were at the6d,7d,8d of estrus, respectively. We found that recipients at the6d of estrus matched to early blastocysts or compacted morula, the7d estrus matched to early blastocysts or blastocysts got the highest pregnancy rate, and8d estrus cow were more suitable for the expanded blastocyst or hatched blastocyst.3) We found that transfer of one embryo resulted in lower pregnant rate than transfer of two or three embryos (57.9%vs71.4vs69.8%%, P<0.05). However, transfer of2or3embryos lead to a high rate of early stage abortion (P<0.05) and low term development than transfer of one embryo(25.0%vs29.5%vs45.4%, P<0.05).4) The vitrification freezing-thawing protocol resulted in almost 100%embryo recovery of hatched blastocyst, and got the highest pregnant after transplantation.3、The production of fat-1transgenic cow by modified SCNT protocolsThe fat-1gene was isolated from Nematodes, humanized and constructed an expression vector. The expression vectors were then confected to a primary bovine fetal fibroblast cells and the transgenic positive cells were used as donor cells for nuclear transfer. The transgenic blastocysts were transferred to estrus recipient cow one fat-1transgenic calf, named ZK002was delivered. The analysis of ZK002indicated that the fat-1gene was successfully integrated into the genome. The fatty acid analyses of ear skin tissue revealed that the content of all ω-3PUFAs was obviously increased, and the content of ω-6PUFAs was significantly decreased. The transgenic cow was with healthy reproduction and naturally delivered a healthy calf. The milk also contained high level ω-3PUAFs, and the ratio between ω-6and ω-3was approximately to1.0. |
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