不同激活方法对水牛ICSI介导转基因效果的影响

Effects of Different Activation Treatments on ICSI-Mediated Gene Transfer in Water Buffalo

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。

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 p.mol/L Ionomycin （Ion） for 5 minutes, and then were divided into three groups： The CHXSh group （incubated in 10 p.g/mL cycloheximide, CHX, for 5 hours）, the CHX3h- DMAP2h group （incubated in 10 p.g/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 CHXSh 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〈O.05）. The rate of intact sperm head in either CHXSh 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 CHXSh 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.