Genome Analysis Laboratory, Animal Genetics Division, ICAR-Indian Veterinary Research Institute, Izatnagar, Bareilly, 243122, UP, India.
AgriGenome Labs Pvt. Ltd., Kakkanad, Cochin, Kerala, India.
Sci Rep. 2018 Feb 16;8(1):3169. doi: 10.1038/s41598-018-21558-9.
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.
由于目前转基因技术方法的成功率有限，转基因家畜的生产受到限制。睾丸介导的基因转移（TMGT）是一种新出现的方法，在产生转基因小鼠方面显示出很高的成功率。在本研究中，我们报告了一种新开发的电穿孔辅助 TMGT 方法，用于生产转基因山羊。首先对转基因的注射体积和浓度进行了标准化，然后对体外电穿孔条件进行了优化。通过将转基因构建体（pIRES2-EGFP；增强型绿色荧光蛋白）注射到睾丸间质中，然后进行电穿孔，在体内进行实验。免疫组织化学、实时定量 PCR（qPCR）和 Western blot 分析证实了转基因成功转移到生精小管和睾丸细胞中。此外，还评估了转基因在精子中的染色体整合及其在 d60 和 d120 后的表达。我们的方案既没有改变精子的特征，也没有改变精子的受精能力。使用转基因精子进行体外受精产生了荧光胚胎。最后，通过自然交配，一只预建系种羊生产了一只转基因小羊。本研究首次成功报告了一种电穿孔辅助 TMGT 方法用于山羊的基因转移。
