A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors
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ORIGINAL PAPER
A new method to efficiently produce transgenic embryos and mice from low-titer lentiviral vectors Kai Miao • Min Guo • Lei An • Xiao Ling Xu Han Wu • Dong Wang • Zhong Hong Wu • Jian Hui Tian
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Received: 14 April 2010 / Accepted: 25 May 2010 / Published online: 29 June 2010 Ó Springer Science+Business Media B.V. 2010
Abstract 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 onecell embryos that were shrunk with a highly concentrated sucrose solution. The survival rate of embryos was greater than 98% using this micromanipulation
Zhong Hong Wu and Jian Hui Tian contributed equally to this work. K. Miao M. Guo L. An X. L. Xu Z. H. Wu J. H. Tian (&) Key Laboratory of Animal Genetics and Breeding of the Ministry of Agriculture, College of Animal Sciences and Technology, China Agricultural University, 100193 Beijing, People’s Republic of China e-mail: [email protected] H. Wu State Key Laboratory of Integrated Management of Pest Insects and Rodents, Institute of Zoology, The Chinese Academy of Sciences, 100101 Beijing, People’s Republic of China
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 9 108 I.U./ml. Even when a low titer of lentivirus (2 9 106 I.U./ml) was used, 53.26% and 40.85% transgenic embryos were obtained after twocell 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. Keywords Lentiviral vectors Transgenic embryos Low titer Mouse
Introduction D. Wang Key Laboratory for Farm Animal Genetic Resources and Utilization of Ministry of Agriculture of China, Institute of Animal Science, Chinese Academy of Agricultural Sciences, 100193 Beijing, People’s Republic of China
Transgenic animals provide an outstanding in vivo model for biomedical research because they can be used to generate preclinical models of human
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diseases and to develop gene therapy str
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