大珠母贝精子介导外源基因转移研究

Electroporation of Sperm to Introduce Foreign DNA into the Genome of Pinctada maxima (Jameson)

将大珠母贝(PinctadamaximaJameson)精子与“全鱼”GH基因重组体pCAgcGH和pCAgcGHc的线性DNA混合,温育30min,经6次、27、10kV脉冲电处理后,与卵子受精,得到若干贝苗。从贝苗中提取DNA,经PCR扩增和Southernblot分子杂交表明,部分受体带有外源基因,当与精子温育的外源基因浓度分别为2μg/mL,6μg/mL及18μg/mL时,相应贝苗携带外源基因比率分别为56%,20%和50%。即在此范围内,基因转移的阳性率与外源基因的浓度呈正相关。

Gene transfer was investigated in marine molluscs via electroporated sperm.Sperm of P.maxima (J.) was incubated with linear “all fish” growth hormone gene (pCAgcGH and pCAgcGHc) for 30min.Then,mature eggs were in vitro fertilized with the sperm cells treated with electroporation at 10kV and 2 7 pulses for six cycles.DNA was extracted from spat and analyzed by PCR and southern blot.The results indicated that the foreign DNA had been transferred into the genome of experimental molluscs.The transgenetic ration was 5.6%,20% and 50% when 2μg/mL,6μg/mL and 18μg/mL of foreign DNA was used,respectively.It is suggested that the transferred efficiency is correlated with the amount of the foreign DNA.