山羊精子介导法转染外源基因影响因素及最适条件的研究

Study on the Influencing Factors of Transfection Efficiency and the Optimum Conditions for Sperm-mediated Gene Transfer in Goats

探讨山羊精子与外源DNA共孵育转染外源DNA效率的影响因素,优化精子转染程序。用DIG免疫组化方法检测和比较精子转染效率,因素为精子洗涤程度、转染缓冲液、精子活力、BSA、肝素、异种动物精浆等。离心洗涤3次以内的山羊精子DNaseⅠ消化前、后阳性率随洗涤次数增加而显著增加(P<0.05),而离心3次以上的转染效率略有下降;以mDM为共孵育缓冲体系可获得较高的转染效率;活力为0.55的精子转基因阳性率显著低于活力为0.9的精子(消化前、后阳性率分别为35.4±2.9%和21.8±5.3%vs 63.5±7.2%和50.3±2.8%,P<0.05);共孵育体系中异种动物精浆可显著降低转染效率(P<0.01),甚至与肝素可置换出已结合和内化转运到精子内的外源DNA,而共孵育体系中的BSA可抑制精子内化外源DNA。山羊共孵育法转染外源基因最适转染条件为mDM缓冲液洗涤3次后上浮收集高活力精子进行转染。

The possible factors affecting the efficiency of sperm taking up exogenoius DNA by co-incubation were investigated to optimize the conditions and procedure of sperm-mediated gene transfer in goats.The transgenic positive goat sperms were detected by in situ hybridization by DIG-labeled mono-cloned antibody.The following influencing factors were investigated:sperm-washing times,the types of transfection buffer,sperm viability,bovine serum albumin,heparin and seminal plasma of different type of animal.The positive transfection rates of sperms before and after digestion with DNase I were significantly increased by increasing the washing times of sperm centrifuged for washing within three times,but after that it decreased.The highest transfection rates could be obtained in mDM co-incubating buffer system.The transfection rates of sperms which viability was 0.55 were significantly lower than that of which viability was 0.99(The transfection rates of sperms before and after digestion with DNase I were 35.4±2.9%and 21.8±5.3%vs 63.5±7.2%and 50.3±2.8%,respectively,P<0.05).And the transfection rates of sperm could be significantly decreased by foreign seminal plasma added in co-incubating buffer system(P<0.01 )and the exogenous DNA molecules bound and internalized into sperm could be replaced by heparin and seminal plasma from goat or pig.Meanwhile,BSA added in co-incubating buffer system could restrain the internalizing process of exogenous DNA molecules into sperm.The optimized conditions of co-incubating sperm with exogenous DNA is:washing sperm 3 times with mDM buffer and using the swimming-up sperm for transfection.