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本研究探讨了不同激活方法对水牛ICSI介导转基因效果的影响。水牛体外成熟卵进行ICSI介导转基因(ICSI-mediated gene transfer,ICSI-Tr)操作后,先用5μmol/L的离子霉素(ionomycin,Ion)激活处理5min,然后分成3组,再分别用10μg/mL放线菌酮(cycloheximide,CHX)培养5h(CHX5h组)、10μg/mLCHX培养3h后再用2mmol/L的二甲氨基嘌呤(6-DMAP)培养2h(CHX3h-DMAP2h组)或用培养液(CM)培养3h后再用2mmol/L6-DMAP培养2h(CM3h-DMAP2h组)。结果显示,CHX5h组的分裂率、早期胚胎基因表达率和囊胚发育率最高,且显著高于CM3h-DMAP2h组(66.7%比49.5%,p<0.05;66.7%比40.0%,p<0.01;22.2%比9.9%,p<0.05)。ICSI18h后检查原核形成率,发现CHX5h和CHX3h-DMAP2h处理组的完整精子头百分率显著低于CM3h-DMAP2h处理组(p<0.05),2原核(pronucleus,PN)百分率则显著提高(42.4%比23.8%,p<0.05;44.6%比23.8%,p<0.01)。以上结果表明,在水牛ICSI介导转基因时,Ion联合CHX较Ion联合6-DMAP激活重构胚效果好。
Abstract：This study investigated effects of different activation treatments on efficiency of ICSI-Mediated gene transfer(ICSI-Tr) in water buffalo.After manipulated by ICSI-Mediated Gene Transfer(ICSI-Tr) in vitro,the matured buffalo oocytes were firstly activated by 5 μmol/L Ionomycin(Ion) for 5 minutes,and then were divided into three groups:The CHX5h group(incubated in 10 μg/mL cycloheximide,CHX,for 5 hours),the CHX3hDMAP2h group(incubated in 10 μg/mL cycloheximide,CHX,for 3 hours,followed by 2 mmol/L 6-DMAP incubation for 2 hours),the CM3h-DAMP2h group(incubated in culture medium,CM,for 3 hours,followed by incubation in 2 mmol/L 6-DMAP for 2 hours).The results showed that the cleavage rate,EGFP expressing rate and blastocyst development rate of CHX5h group were the highest and significantly higher than CM3h-DMAP2h group(66.7% vs.49.5%,p<0.05;66.7% vs.40.0%,p<0.01;22.2% vs.9.9%,p<0.05).The rate of intact sperm head in either CHX5h or CHX3h-DMAP2h treatment group with ICSI 18 h was significantly lower than the CM3h-DMAP2h Group(p<0.05) after checking the prokaryotic formation rate.For the pronucleus(PN) formation rate,the CHX5h group and CHX3h-DMAP2h group were significantly higher than CM3h-DMAP2h group(42.4% vs.23.8%,p<0.05;44.6% vs.23.8%,p<0.01).These results indicated that the activation effect of reconstructed embryos in Ion combined with CHX was better than that in Ion combined with 6-DMAP ICSI-Mediated during gene transfer(ICSI-Tr) in water buffalo.
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基本信息:
中图分类号:S823
引用信息:
[1]陈自洪,韦珏,孟凡丽,等.不同激活方法对水牛ICSI介导转基因效果的影响[J].基因组学与应用生物学,2012,31(04):355-359.
基金信息:
国家高技术研究发展计划(863)项目(2011AA100607);; 国家转基因重大专项(2010ZX08007-003);; 国家自然科学基金(30960251);; 桂科自0991007Z;; 南宁科201003070G共同资助
2012-08-28
2012-08-28