电脉冲作用将外源基因导入稀有(鱼句)鲫精子的研究

将稀有鲫 (Gobiocyprisrarus)精子与重组质粒pCAhLFc线性DNA混合温育 ,经电脉冲处理后与卵子受精 ,孵化出苗。从鱼苗中提取DNA ,经PCR检测 ,2 5 .5 %～ 6 6 .7%鱼苗带有外源基因。在显微镜下观察经电脉冲处理过的精子 ,发现其活力有不同程度下降 ,受精率也有不同程度下降 ,说明不同的电脉冲条件对精子有不同程度的损害作用。精子与外源DNA混合温育 ,经电脉冲处理后 ,用DNA外切酶消化后 ,提取精子DNA ,经PCR检测 ,仍有阳性电泳带 ,证明电脉冲可以促使稀有鲫精子摄入外源基因

Introduction of Foreign DNA into the Genome of Rare Minnow by Sperm Electroporation

Gene transfer was investigated in rare minnow via electroporated sperm. The sperm of the fish mixed with linear DNA (pCAhLFc) was electroporated. Then mature eggs were in vitro fertilized with these sperm cells. The DNA was extracted from the fries and analyzed by PCR. The percentage of fries with foreign genes varied between 25.5% and 66.7%. The observation of the electroporated sperm under microscope indicates that both the vigor of sperms and the successful ratio of the fertilization diminish, indicating that the electroporation with various parameters can hurt the sperm on different extent. The sperm cells electroporated with foreign genes, then was treated by DNA, and the DNA was extracted and was analyzed by PCR. It was found that foreign gene still exists, which approves that the electroporation can force the foreign gene enter the sperm of rare minnow.