Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa
Abstract
Transgenic technologies conventionally rely on the oocyte as a substrate for genetic modification. Owing to their accessibility, however, male germ cells, including mature sperm, have material advantages for use in transgenesis. Here we have exploited lentiviruses to generate transgenic animals via the male germline. When pseudotyped lentiviral vectors encoding green fluorescent protein (GFP) were incubated with mouse spermatozoa, these sperm were highly successful in producing transgenics. Lentivirally transduced mouse spermatozoa were used in in vitro fertilization (IVF) studies, and when followed by embryo transfer, ≥42% of founders were found to be transgenic for GFP. Inverse PCR strategy for integration site analysis demonstrated integration of at least 1 or 2 copies of GFP in the transgenics, mapping to different chromosomes. GFP expression was detected in a wide range of murine tissues, including testis and the transgene was stably transmitted to a third generation of transgenic animals. This relatively simple, yet highly efficient, technique for generating transgenic animals by transducing spermatozoa with lentiviral vectors in vitro is a powerful tool for the study of fertilization/preimplantation development, vertical viral gene transmission, gene function and regulation, and epigenetic inheritance.
Additional Information
© 2014 FASEB. Received for publication May 31, 2013. Accepted for publication October 3, 2013. T he authors thank Malcolm Parker and Ilpo Huhtaniemi (Imperial College London) for critically reading the manuscript. The authors also thank Aura Keeter, Natasha Bouey, and Joaquin Gutierrez (California Institute of Technology), and Ihsan Isa (Imperial College London), for their dedicated technical assistance; and Naveenan Navaratnam and Geok Chin Tan (Imperial College London) for helpful discussion and support. The authors thank Amy Cutler and Zoe Webster (Transgenic and Embryonic Stem Cell Laboratory, Medical Research Council–Clinical Sciences Centre, London, UK) for assistance with IVF followed by embryo transfer to obtain transgenic animals, and their maintenance and welfare. The authors thank Luigi Naldini [San Raffaele Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan, Italy] for providing the PGK-GFP encoded lentiviral vector and Rusty Landsford (California Institute of Technology, Pasadena, CA, USA) for the vectors containing H2Bcherry reporters. This work was supported by Atazoa Ltd. (London, UK) and by the Genesis Research Trust (previously known as the Institute of Obstetrics and Gynaecology Trust; London, UK). R.S. was supported by a Case Ph.D. studentship from the UK Biotechnology and Biological Resources Research Council. Author contributions: A.C. and C.R. designed and performed experiments and analyzed the data; R.S. performed experiments; C.C., S.E.F., A.T., N.D., and R.W. contributed reagents, lent support and advice on experiments, and contributed to the writing and editing of the manuscript; A.C. and C.R. wrote the manuscript. R.W. and C.R. are joint senior authors. Conflicts of interest: C.R., N.D., and R.W. are directors of a commercial enterprise, Atazoa Ltd., which hopes to exploit novel techniques for animal transgenesis. A.C. is a former employee of Atazoa Ltd.Additional details
- Eprint ID
- 44460
- Resolver ID
- CaltechAUTHORS:20140324-110356014
- Atazoa Ltd.
- Genesis Research Trust
- Biotechnology and Biological Sciences Research Council (BBSRC)
- Created
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2014-03-26Created from EPrint's datestamp field
- Updated
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2021-11-10Created from EPrint's last_modified field