Abstract
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.
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References
Gordon JW, Ruddle FH (1982) Germ line transmission in transgenic mice. Prog Clin Biol Res 85(Pt B):111–124
Paris MC, Snow M, Cox SL, Shaw JM (2004) Xenotransplantation: a tool for reproductive biology and animal conservation? Theriogenology 61:277–291
Prather RS (2007) Targeted genetic modification: xenotransplantation and beyond. Cloning Stem Cells 9:17–20
Nagano M, Brinster CJ, Orwig KE et al (2001) Transgenic mice produced by retroviral transduction of male germ-line stem cells. Proc Natl Acad Sci U S A 98:13090–13095
Dhup S, Majumdar SS (2008) Transgenesis via permanent integration of genes in repopulating spermatogonial cells in vivo. Nat Methods 5:601–603
Sehgal L, Thorat R, Khapare N et al (2011) Lentiviral mediated transgenesis by in vivo manipulation of spermatogonial stem cells. PLoS One 6:e21975
Lavitrano M, Busnelli M, Cerrito MG et al (2006) Sperm-mediated gene transfer. Reprod Fertil Dev 18:19–23
Howe SJ, Chandrashekran A (2012) Vector systems for prenatal gene therapy: principles of retrovirus vector design and production. Methods Mol Biol 891:85–107. doi:10.1007/978-1-61779-873-3_5
Lavial F, Bessonnard S, Ohnishi Y et al (2012) Bmi1 facilitates primitive endoderm formation by stabilizing Gata6 during early mouse development. Genes Dev 26:1445–1458. doi:10.1101/gad.188193.112, gad.188193.112 [pii]
Witney TH, Carroll L, Alam IS et al (2014) A novel radiotracer to image glycogen metabolism in tumors by positron emission tomography. Cancer Res 74:1319–1328. doi:10.1158/0008-5472.CAN-13-2768, 74/5/1319 [pii]
Tan GC, Chan E, Molnar A et al (2014) 5′ isomiR variation is of functional and evolutionary importance. Nucleic Acids Res 42:9424–9435. doi:10.1093/nar/gku656, gku656 [pii]
Chandrashekran A, Isa I, Dudhia J, Thrasher AJ et al (2014) Lentiviral vector transduction of spermatozoa as a tool for the study of early development. FEBS Open Bio 4:266–275. doi:10.1016/j.fob.2014.02.008, S2211-5463(14)00019-9 [pii]
Chandrashekran A, Sarkar R, Thrasher A et al (2014) Efficient generation of transgenic mice by lentivirus-mediated modification of spermatozoa. FASEB J 28:569–576. doi:10.1096/fj.13-233999, fj.13-233999 [pii]
Chandrashekran A, Gordon MY, Casimir C (2004) Targeted retroviral transduction of c-kit + hematopoietic cells using novel ligand display technology. Blood 104:2697–2703
Chandrashekran A, Gordon MY, Darling D, Farzaneh F, Casimir C (2004) Growth factor displayed on the surface of retroviral particles without manipulation of envelope proteins is biologically active and can enhance transduction. J Gene Med 6:1189–1196
Zufferey R, Dull T, Mandel RJ et al (1998) Self-inactivating lentivirus vector for safe and efficient in vivo gene delivery. J Virol 72:9873–9880
Ailles LE, Naldini L (2002) HIV-1-derived lentiviral vectors. Curr Top Microbiol Immunol 261:31–52
Pan D, Gunther R, Duan W et al (2002) Biodistribution and toxicity studies of VSVG-pseudotyped lentiviral vector after intravenous administration in mice with the observation of in vivo transduction of bone marrow. Mol Ther 6:19–29
Landazuri N, Le Doux JM (2006) Complexation with chondroitin sulfate C and Polybrene rapidly purifies retrovirus from inhibitors of transduction and substantially enhances gene transfer. Biotechnol Bioeng 93:146–158
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Chandrashekran, A., Casimir, C., Dibb, N., Readhead, C., Winston, R. (2016). Generating Transgenic Mice by Lentiviral Transduction of Spermatozoa Followed by In Vitro Fertilization and Embryo Transfer. In: Federico, M. (eds) Lentiviral Vectors and Exosomes as Gene and Protein Delivery Tools. Methods in Molecular Biology, vol 1448. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3753-0_8
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DOI: https://doi.org/10.1007/978-1-4939-3753-0_8
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Publisher Name: Humana Press, New York, NY
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Online ISBN: 978-1-4939-3753-0
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