慢病毒载体法在制备转基因小鼠模型中的应用

Application of lentiviral vectors in establishment of transgenic mice models

目的改进慢病毒载体法制备转基因小鼠的操作技术,分别从病毒浓缩、受精卵显微注射进针点和如何提高病毒注射效率等方面探讨慢病毒显微注射技术的最佳方案。方法超速离心获得浓缩慢病毒,通过卵周隙显微注射技术感染小鼠受精卵,比较不同注射点显微注射后受精卵存活率及发育情况。结果受精卵1点和5点作为注射点的卵周隙注射组可有效避免注射针与卵膜的直接碰触,减少对受精卵的损伤,胚胎存活率较常规中轴3点作为注射点组显著提高(P<0.01)。适当的病毒滴度和注入量、操作细节、注射针口径等均可影响注射后受精卵存活率和感染率。结论卵周隙显微注射过程中选择适当的注射点以及部分操作的改进,可显著提高受精卵的存活率,为提高转基因小鼠模型的制备效率提供参考。

Purpose To improve the skills of subzonal injection by lentiviral vectors and to find the most suitable way of lentiviral microinjection in terms of lentiviruses concentration,injection point and efficiency.Methods High concentrated lentiviruses were harvested by ultracentrifugation.The lentiviruses were introduced into fertilized eggs by pushing the micropipette tip through the zona layer into the perivitelline space.The survival rates of fertilized eggs were analyzed and compared after injected through three different points.Results The survival rates of manipulated eggs were significantly increased through the 1 and 3 points injection compared with central axis 3 points injection（P〈0.01）,which avoided harming the embryo cell membrane.Lentivirus titer,manipulation and micropipette were the influencing factors in this operation.Conclusions The suitable injection point and improved manipulation in perivitelline space microinjection can significantly increase the survival rate of fertilized eggs and promote the efficiency of establishing transgenic mice by lentiviral vectors.