Intratesticular injection followed by electroporation allows gene transfer in caprine spermatogenic cells

The production of transgenic livestock is constrained due to the limited success of currently available methods for transgenesis. Testis mediated gene transfer (TMGT) is an emerging method that shows a high success rate in generating transgenic mice. In this study, we report a newly developed protoc...

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Bibliographic Details
Published in:Scientific reports 2018-02, Vol.8 (1), p.3169-11, Article 3169
Main Authors: Pramod, R. Kumar, Mitra, Abhijit
Format: Article
Language:English
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Electroporation
Embryos
Gene transfer
Green fluorescent protein
Humanities and Social Sciences
Immunohistochemistry
In vitro fertilization
Livestock
multidisciplinary
Rodents
Science
Science (multidisciplinary)
Sperm
Testes
Transgenic mice
Tubules
Western blotting
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Summary:The production of transgenic livestock is constrained due to the limited success of currently available methods for transgenesis. Testis mediated gene transfer (TMGT) is an emerging method that shows a high success rate in generating transgenic mice. In this study, we report a newly developed protocol for electroporation-aided TMGT to produce a transgenic goat. The injectable volume and concentration of the transgene were first standardized, and then electroporation conditions were optimized in vitro . In vivo experiments were performed by injecting a transgenic construct ( pIRES2-EGFP ; enhanced green fluorescent protein) into the testicular interstitium followed by electroporation. Immunohistochemistry, quantitative real-time PCR (qPCR) and western blotting analyses confirmed the successful transfer of the transgene into seminiferous tubules and testicular cells. Furthermore, chromosomal integration of the transgene and its expression in sperm were evaluated d60 and d120 post-electroporation. Our protocol neither altered the seminal characteristics nor the fertilization capacity of the sperm cells. In vitro fertilization using transgenic sperm generated fluorescent embryos. Finally, natural mating of a pre-founder buck produced a transgenic baby goat. The present study demonstrates the first successful report of an electroporation-aided TMGT method for gene transfer in goats.
ISSN:2045-2322
2045-2322
DOI:10.1038/s41598-018-21558-9