Abstract
More than 20 years have passed since the advent of genetic manipulation of the mouse germline using cultures of pluripotent embryonic stem cells. Still, despite remarkable successes in the mouse, the application of stem cell cultures for transgenesis in other mammalian species has been comparatively nonexistent. By focusing on the laboratory rat as a widely popular model species in science, this chapter highlights several advantages of the spermatogonium as an alternative type of germline stem cell for transgenesis. Protocols for isolating, propagating, genetically modifying, and determining the germline transmission rates of spermatogonial cultures for the production of transgenic rats are introduced in detail. Although the full potential of spermatogonia has yet to be realized in animal genetics, this chapter illustrates how their application as novel germline vectors would open new doors to advance transgenic technology. Most notably, gene manipulations directly in the spermatogonium simplify production of germline founders while bypassing the intermediate production of chimeric progeny using micromanipulated embryos. Once experimental conditions for producing genetically modified animals using spermatogonial cultures are optimized, the approach holds the potential to facilitate targeted germline modifications by gene replacement, gene-insertion and/or restriction endonuclease technologies in a diversity of mammalian species.
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Notes
- 1.
Safety Information: Lentiviral vectors are considered biohazardous reagents. Their use for studies in animals warrants an approved safety plan certified by both the parent research institution’s Environmental Health and Safety program, and the Institutional Animal Care and Use Committee (IACUC). Personal protection equipment (gloves; lab coat; safety glasses) should be worn when working with lentiviral vectors to prevent exposure to the researcher. The preparation, use and disposal of lentiviral vectors should be restricted to certified biosafety cabinets to prevent exposure to the researcher and to others.
- 2.
Safety Information: Busulfan is considered a biohazardous compound. Its use for studies in animals warrants an approved safety plan certified by both the parent research institution’s Environmental Health and Safety program, and the Institutional Animal Care and Use Committee (IACUC). Personal protection equipment (doubled gloves; lab coat; safety glasses; ventilation mask) should be worn when working with busulfan to prevent any type of exposure to the researcher. The preparation, use and disposal of busulfan should all be restricted to certified chemical and/or biosafety cabinets to prevent exposure to the researcher and others.
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Acknowledgments
Studies to establish the methodology reported herein was supported by NIH grants R21RR023958 from the National Center for Research Resources and RO1HD036022 from the National Institute of Child Health and Human Development, and by the Cecil H. & Ida Green Center for Reproductive Biology Sciences at the University of Texas Southwestern Medical Center in Dallas.
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Chapman, K.M., Saidley-Alsaadi, D., Syvyk, A.E., Shirley, J.R., Thompson, L.M., Hamra, F.K. (2011). Rat Spermatogonial Stem Cell-Mediated Gene Transfer. In: Pease, S., Saunders, T. (eds) Advanced Protocols for Animal Transgenesis. Springer Protocols Handbooks. Springer, Berlin, Heidelberg. https://doi.org/10.1007/978-3-642-20792-1_12
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DOI: https://doi.org/10.1007/978-3-642-20792-1_12
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