一种改进的显微注射用DNA片段的纯化方法

目的显微注射用DNA的纯度是影响转基因动物制备成功与否的重要因素,本文建立一种可行的适用于普通实验室的纯化DNA方法,替代普遍使用的试剂盒纯化方法。方法分别使用酚-氯仿多次抽提法及常规的凝胶提取试剂盒纯化含有蚓激酶基因的DNA片段,通过显微注射技术将纯化的DNA片段导入小鼠受精卵的原核,制备转基因小鼠。根据转基因实验的结果对两种方法进行比较。结果使用两种方法纯化DNA均能获得转基因小鼠。在DNA纯度及注射卵的存活率上,两种方法无明显差别;在移植卵的出生率及转基因阳性率上,抽提法优于试剂盒法。结论本实验建立的抽提方法可以替代试剂盒方法纯化显微注射用DNA片段,在降低实验成本、简化实验条件及提高转基因阳性率方面具有优势。

An Improved Method of Purification of DNA Fragment for Microinjection

Objective The purity of DNA for microinjection is a crucial factor for preparing transgenic animals.The aim of this study is to develop an improved DNA purifying method for preparation of DNA fragments in high purity,feasible for routine transgenic animal research work.Methods DNA fragments containing lumbrokinase gene were extracted and purified by phenol-chloroform repeated extraction method and routine gel extraction kit.The purified DNA was microinjected into pro-nucleus of mice fertilized eggs and the injected living eggs were transplanted into the oviducts of pseudo-pregnant mice.The offspring after transplantation were identified by Southern blot.The two purifying methods were compared according to the results of transgenic experiments.Results Transgenic mice could be obtained with DNA purified by both two methods.There was no notable difference in DNA purity and viability of injected eggs,but higher birth rate of transplanted eggs and transgenic positive rate were obtained by the extraction method than those obtained by the kit method.Conclusion Our findings indicate that the extraction method can be used in purifying DNA for microinjection,replacing the kit method,with advantages such as lower cost,simplified experimental procedure,and improved transgenic positive rate in preparation of transgenic animals.