A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors

文献类型： 外文期刊

第一作者： Miao, Kai

作者： Miao, Kai;Guo, Min;An, Lei;Xu, Xiao Ling;Wu, Zhong Hong;Tian, Jian Hui;Wu, Han;Wang, Dong

作者机构：

关键词： mice.;Lentiviral vectors;Transgenic embryos;Low titer;Internet resource.

期刊名称：TRANSGENIC RESEARCH （ 影响因子：2.788； 五年影响因子：2.377 ）

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收录情况： SCI

摘要： Vector injection into the perivitelline space has emerged as the standard delivery method to transduce lentivirus to mammalian oocytes or one-cell embryos, but its application is limited by the need for high titers of lentivirus. Herein we developed a new method by using a Piezo impact micro-manipulator for injecting low titer of lentivirus into the subzonal space of two-cell embryos or the perivitelline space of one-cell embryos that were shrunk with a highly concentrated sucrose solution. The survival rate of embryos was greater than 98% using this micromanipulation strategy, which was increased compared to the normal one-cell embryo injection method. More than 90% of injected embryos were GFP positive after subzonal injection of a lentivirus vector carrying the GFP gene with titers of 2 c 10i I.U./ml. Even when a low titer of lentivirus (2 c 10e I.U./ml) was used, 53.26% and 40.85% transgenic embryos were obtained after two-cell embryonic injection and one-cell sucrose treated embryonic injection, respectively. The GFP-positive rates were also greater than in the conventional method of injecting one-cell embryos (25.39%). In addition, blastocysts from the two-cell embryo injection group displayed stronger GFP fluorescence than the one-cell embryo injection groups treated with or without the sucrose solution. Increased expression of GFP suggests that the embryos obtained from this injection method have higher exogenous gene expression levels compared to previous methods. Therefore, in contrast with the traditional injection method, we have demonstrated a simplified and efficient lentivirus-mediated gene transfer method based on a low-titer virus preparation.

分类号： R394

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