Abstract
Lentiviruses are highly efficient vehicles for delivering genes into cells. They readily transduce primary and immortalized cells in vivo and in vitro. Genes delivered by lentiviruses are incorporated and replicated as part of their host genome and therefore offer a powerful tool for creation of stable cell lines and transgenic animals. However, the zona pellucida surrounding the fertilized eggs acts as a barrier and hinders lentiviral transduction of embryos. Here, we utilize a laser, typically used to perforate the zona pellucida for in vitro fertilization, to permeabilize the zona for lentiviral gene delivery. A single hole in the zona is sufficient for the lentivirus to gain access to fertilized eggs without the need for microinjection for en masse gene delivery. Embryos generated by this method elicit no damage and can develop to term for creation of transgenic animals.
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Acknowledgements
This research was supported by the Intramural Research Program of the National Institute of Health (NIH), National Institute of Environmental Health Sciences (NIEHS). We are grateful to Dr. Franco Demayo, Dr. Ronald Cannon, Terry Blankenship, and Dr. Edward (Mitch) Eddy for critical reading of the manuscript and helpful advice. We would also like to acknowledge and thank Dr. Bernd Gloss, the Knockout core, the Flow Cytometry Facility, the Fluorescent Microscopy, and Imaging Center and the Comparative Medicine Branch facilities of the NIEHS for their technical contributions. We would like to thank Mr. David Goulding from the Comparative Medicine Branch; Mr. Steve McCaw, and Ms. Lois Wyrick of the Imaging Center at the NIEHS for providing us with photographs of our instruments and illustrations for the figures.
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Martin, N.P., Myers, P., Goulding, E. et al. En masse lentiviral gene delivery to mouse fertilized eggs via laser perforation of zona pellucida. Transgenic Res 27, 39–49 (2018). https://doi.org/10.1007/s11248-017-0056-8
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DOI: https://doi.org/10.1007/s11248-017-0056-8