Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa

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Title
Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa
Author(s)
Bo Woong Sim; Jae Jin Cha; Bong Seok SongJi Su KimSeung-Bin Yoon; Seon A Choi; Kang Jin JeongYoung-Hyun KimJae Won Huh; Sang Rae Lee; Sang-Hyun Kim; C S Lee; Sun-Uk Kim; Kyu Tae Chang
Bibliographic Citation
Molecular Reproduction and Development, vol. 80, pp. 233-241
Publication Year
2013
Abstract
Many methods for efficient production of transgenic animals for biomedical research have been developed. Despite great improvements in transgenesis rates resulting from the use of intracytoplasmic sperm injection (ICSI), the ICSI-based sperm-mediated gene-transfer (iSMGT) technique is still not optimal in terms of sperm permeabilization efficiency and subsequent development. Here, we demonstrate that streptolysin-O (SLO) can efficiently permeabilize mouse spermatozoa, leading to improved developmental competence and high transgenesis rates in iSMGT embryos and pups. In particular, the most efficient production of iSMGT-transgenic embryos resulted from pretreatment with 5U/ml SLO for 30min and co-incubation with 1.0ng/μl of an EGFP expression vector. By incubating spermatozoa with Cy-3-labelled DNA, we found that fluorescence intensity was prominently detected in the head region of SLO-treated spermatozoa. In addition, blastocyst development rate and blastomere survival were greatly improved by iSMGT using SLO-treated spermatozoa (iSMGT-SLO) as compared to freeze-thawed spermatozoa. Consistent with this, a high proportion of transgenic offspring was obtained by iSMGT-SLO after transfer into foster mothers, reaching 10.6% of the number of oocytes used (42.3% among pups). Together with successful germline transmission of transgenes in all founders analyzed, our data strongly suggest that SLO makes spermatozoa amenable to exogenous DNA uptake, and that the iSMGT-SLO technique is an efficient method for production of transgenic animals for biomedical research.
ISSN
1040-452X
Publisher
Wiley
DOI
http://dx.doi.org/10.1002/mrd.22158
Type
Article
Appears in Collections:
Ochang Branch Institute > Division of National Bio-Infrastructure > Futuristic Animal Resource & Research Center > 1. Journal Articles
Jeonbuk Branch Institute > Primate Resources Center > 1. Journal Articles
Ochang Branch Institute > Division of National Bio-Infrastructure > National Primate Research Center > 1. Journal Articles
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