Efficient production of transgenic mice by intracytoplasmic injection of streptolysin-O-treated spermatozoa.
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 5 U/ml SLO for 30 min and co-incubation with 1.0 ng/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.
National Primate Research Center, Korea Research Institute of Bioscience and Biotechnology, Chungcheongbuk-do, Republic of Korea.
Bo-Woong
BW
Sim
Jae-Jin
JJ
Cha
Bong-Seok
BS
Song
Ji-Su
JS
Kim
Seung-Bin
SB
Yoon
Seon-A
SA
Choi
Kang-Jin
KJ
Jeong
Young-Hyun
YH
Kim
Jae-Won
JW
Huh
Sang-Rae
SR
Lee
Sang-Hyun
SH
Kim
Chul-Sang
CS
Lee
Sun-Uk
SU
Kim
Kyu-Tae
KT
Chang
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000494:pharmacology; D001709:Biotechnology / Q000379:methods*; D047108:Embryonic Development / Q000187:drug effects; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000196:embryology*; D020554:Sperm Injections, Intracytoplasmic / Q000379:methods / Q000662:veterinary*; D013094:Spermatozoa / Q000187:drug effects / Q000502:physiology*; D013301:Streptolysins / Q000737:chemistry* / Q000494:pharmacology
D001426:Bacterial Proteins; D013301:Streptolysins; C025964:streptolysin O