【作者】 苏建民；

【导师】 张涌；

【作者基本信息】 西北农林科技大学 ， 发育生物学， 2009， 硕士

【摘要】 为了研究有血清培养基SOF-serum和无血清序贯培养基G1.2/G2.2支持牛转基因克隆胚胎发育的能力,本研究系统比较了在SOF-serum(SOF-serum组)和G1.2/G2.2(G1.2/G2.2组)条件下分别培养的牛转基因克隆胚的体外发育情况和胚胎移植后妊娠情况,并研究了两组培养基分别获得的克隆囊胚印迹Igf2的DNA甲基化状况,从而比较培养基SOF-serum和G1.2/G2.2支持牛转基因克隆胚体外发育及移植后体内发育的能力。本研究设计如下:试验1:(1)比较两组的体细胞克隆胚体外发育速度、胚胎外观形态。(2)比较两种培养基的胚胎体外发育情况(卵裂率、囊胚率、囊胚孵化率)。(3)对两组的囊胚分别进行细胞凋亡染色,比较两种培养基的囊胚细胞凋亡情况。(4)对两种培养基的囊胚分别进行差异染色,比较两种培养基对胚胎构成性(ICM/ TE)的影响。试验2:将上述两组第7天的囊胚移植到受体母牛,比较两种培养基对转基因克隆牛妊娠率的影响。试验3:比较两种培养基得到的转基因克隆囊胚的印迹基因Igf2的DNA甲基化状况,探讨不同培养基对胚胎发育相关的印迹基因Igf2甲基化状况的影响,从而进一步验证两种培养基对克隆胚发育的影响。研究结果显示:试验1:(1)两种培养基的体细胞克隆胚胎的发育速度及外观形态有显著区别。G1.2/G2.2组的胚胎在8-细胞期以后发育速度明显快于SOF-serum组的胚胎,SOF-serum组的胚胎桑葚胚致密化及囊胚扩张较G1.2/G2.2组更明显;(2)两组的转基因克隆胚的卵裂率(86.5% vs 88.4%, P > 0.05)差异不显著,但SOF-serum组的囊胚发育率与G1.2/G2.2组有显著性差异(50.3% vs 40.4%,P < 0.05)。另外SOF-serum组第9天囊胚的孵化率显著高于G1.2/G2.2组,差异显著(30.2% vs 21.3%, P < 0.05);(3)两组的转基因克隆囊胚的细胞总数差异不显著(93.2±10.5 vs 88.3±9.8, P > 0.05),但G1.2/G2.2组囊胚的细胞凋亡率显著低于SOF-serum组(8.5±1.2 vs 16.1±1.7, P<0.05);(4)两组囊胚的ICM/ TE差异不显著(0.23±0.03 vs 0.22±0.01);试验2:两组的转基因克隆胚在前40天妊娠率差异不显著(61.1% vs 65.4%, P > 0.05),但60天以后,G1.2/G2.2组的妊娠率显著高于SOF-serum组(46.1% vs 11.1%, P < 0.05),另外G1/G2培养组有23.1%的受体维持妊娠超过120天,但SOF-serum组没有妊娠超过90天;试验3:两组的转基因克隆囊胚的印迹基因Igf2的DNA甲基化状态有差异,两种培养基对胚胎发育相关的印迹基因Igf2的DNA甲基化状况有影响。结果表明,在转基因体细胞核移植研究中,虽然用SOF-serum能获得较高的囊胚率,但其囊胚的细胞凋亡率明显较高,且胚胎移植后其支持胚胎体外发育的能力明显低于无血清序贯培养基G1.2/G2.2。无血清序贯培养基G1.2/G2.2可以用于牛转基因克隆胚胎的体外培养,且胚胎移植后支持体内发育的能力强于常用的SOF-serum培养基,能获得较理想的妊娠率。更多还原

【Abstract】 The objective of this study was to evaluate the effects of single and sequential culture media(SOF-serum, G1.2/G2.2) on the development(in vitro and in vivo) of bovine transgenic cloned embryos and effects on the methylation patterns of Igf2 from blastocysts with different culture media. SCNT embryos were cultured in SOF-serum and G1.2/G2.2, respectively.Experiment 1: (1) We evaluate the effects of SOF-serum and G1.2/G2.2 on the development of rate and shape of bovine cloned embryos.(2) We evaluate the effects of SOF-serum and G1.2/G2.2 on the in vitro development of bovine transgenic cloned embryos(cleavage rate, blastocyst rate and hatched blastocyst rate).(3) We evaluate the effects of SOF-serum and G1.2/G2.2 on the numbers of apoptosis cell in blastocyst.(4) We evaluate the effects of SOF-serum and G1.2/G2.2 on the ratio of ICM to TE cell in blastocysts.Experiment 2: we evaluate the effects of SOF-serum and G1.2/G2.2 on the pregnancy rate.Experiment 3: we evaluate the effects of SOF-serum and G1.2/G2.2 on the methylation patterns of Igf2 from blastocysts.The results show that:Experiment 1: (1) There were different effect on rate of developmenton and embryo shape from two culture media. Embryos from G1.2/G2.2 group after 8-cell stage have a high rate of development than SOF-serum group. But pyknosis of morula and expansion of blastocyst from SOF-serum group were more visible than G1.2/G2.2 group;(2) There was no difference in cleavage rate(86.5% vs 88.4%, P > 0.05), however, SOF-serum group provided the higher blastocyst rate(50.3% vs 40.4%,P < 0.05) and hatched blastocyst rate(31.5% vs 21.3%, P < 0.05).(3) There was no difference in total cell number in blastocyst from two group(93.2±10.5 vs 88.3±9.8, P > 0.05), but numbers of apoptosis cell in blastocyst from SOF-serum group were significantly higher than G1.2/G2.2 group(8.5±1.2 vs 16.1±1.7, P<0.05); (4) The ratio of ICM to TE cell in blastocysts were not different between two groups (0.23±0.03 vs 0.22±0.01);Experiment 2: There was no difference in 40 day pregnancy rate between two groups(46.1% vs 11.1%, P > 0.05), however, after 60 day, G1.2/G2.2 group has a significantly higher pregnancy rate than SOF-serumgroup(46.1% vs 11.1%, P < 0.05).Experiment 3: There was difference in the methylation patterns of Igf2 in blastocysts from both culture media.We conclude that sequential media G1.2/G2.2 could support in vitro and in vivo development of bovine transgenic SCNT embryos, and in vivo developmental competence of embryos was higher than SOF-serum group after embryo tranfer.更多还原