Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer.
Most transgenic technologies rely on the oocyte as a substrate for genetic modification. Transgenics animals are usually generated by the injection of the gene constructs (including lentiviruses encoding gene constructs or modified embryonic stem cells) into the pronucleus of a fertilized egg followed by the transfer of the injected embryos into the uterus of a foster mother. Male germ cells also have potential as templates for transgenic development. We have previously shown that mature sperm can be utilized as template for lentiviral transduction and as such used to generate transgenic mice efficiently with germ line capabilities. We provide here a detailed protocol that is relatively simple, to establish transgenic mice using lentivirally transduced spermatozoa. This protocol employs a well-established lentiviral gene delivery system (usual for somatic cells) delivering a variety of transgenes to be directly used with sperm, and the subsequent use of these modified sperm in in vitro fertilization studies and embryo transfer into foster female mice, for the establishment of transgenic mice.
Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Campus, Institute of Reproductive and Developmental Biology (IRDB), Du Cane Road, London, W12 0NN, UK. a.chandrashekran98@imperial.ac.uk.
Anil
A
Chandrashekran
Department of Natural Sciences, School of Science & Technology, Middlesex University, The Burroughs, London, NW4 4BT, UK.
Colin
C
Casimir
Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Campus, Institute of Reproductive and Developmental Biology (IRDB), Du Cane Road, London, W12 0NN, UK.
Nick
N
Dibb
Translational Imaging Center, University of Southern California, Los Angeles, CA, 90089, USA.
Carol
C
Readhead
Department of Surgery and Cancer, Division of Cancer, Imperial College London, Hammersmith Campus, Institute of Reproductive and Developmental Biology (IRDB), Du Cane Road, London, W12 0NN, UK.
Robert
R
Winston
D000818:Animals; D004624:Embryo Transfer / Q000379:methods; D053595:Embryonic Stem Cells / Q000166:cytology; D005307:Fertilization in Vitro / Q000379:methods; D018014:Gene Transfer Techniques; D005822:Genetic Vectors; D016086:Lentivirus / Q000235:genetics*; D008297:Male; D051379:Mice; D008822:Mice, Transgenic / Q000235:genetics*; D009865:Oocytes / Q000254:growth & development; D013094:Spermatozoa / Q000254:growth & development; D014161:Transduction, Genetic / Q000379:methods*
Gene transfer; Germ line; Lentiviral vectors; Pseudotyping; Spermatozoa; Transgenes; Transgenics