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

Scientific Reports
R Kumar Pramod, Abhijit Mitra

Abstract

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 ...Continue Reading

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Citations

Sep 26, 2019·Frontiers in Genetics·Peter KaldsYulin Chen

Related Concepts

Western Blotting
Embryo
Fertility
Immunohistochemistry
Structure of Interstitial Cell of Leydig
Mice, Transgenic
Sperm Cell
Testis
Plants, Transgenic
Green Fluorescent Proteins

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