基因组编辑被认为是农业和研究的强大工具,增强我们对遗传功能的理解,疾病,和生产力。然而,由于一些挑战,它在水牛方面的进展落后于其他哺乳动物,包括漫长的妊娠期,单胎妊娠,和高昂的筹资成本。在这项研究中,我们的目标是生成MSTN编辑的水牛,以其独特的双肌肉表型而闻名,作为概念的证明。为了实现我们的目标,我们使用体细胞核移植(SCNT)和合子电穿孔(CRISPR-EP)技术。为此,我们首先确定了将RNP复合物导入成纤维细胞的最佳转染方法,并进一步用于SCNT。为此,我们比较了转染,成纤维细胞核转染和脂质转染的裂解效率和细胞活力。乳沟,发现核转染组的转染效率和细胞活力显着高于脂质体转染组(P≤0.05)。使用核转染产生了四个MSTN编辑的菌落,其中发现三个菌落是双等位基因的,一个是单等位基因的。Further,我们比较了疗效,SCNT和合子电穿孔的胚胎发育潜力和随后的妊娠结局。电穿孔组囊胚率明显高于SCNT组(P≤0.05)。然而,合子电穿孔组导致两次妊娠,经证实为MSTN编辑.因为,合子电穿孔不需要与SCNT相关的复杂显微操作技术,它有可能促进水牛等大型家畜的基因改造。本研究为诱导具有实际或生物学意义的遗传交替奠定了基础。
Genome editing is recognized as a powerful tool in agriculture and research, enhancing our understanding of genetic function, diseases, and productivity. However, its progress in buffaloes has lagged behind other mammals due to several challenges, including long gestational periods, single pregnancies, and high raising costs. In this study, we aimed to generate MSTN-edited buffaloes, known for their distinctive double-muscling phenotype, as a proof of concept. To meet our goal, we used somatic cell nuclear transfer (SCNT) and zygotic electroporation (CRISPR-EP) technique. For this, we firstly identified the best
transfection method for introduction of RNP complex into fibroblast which was further used for SCNT. For this, we compared the
transfection, cleavage efficiency and cell viability of nucleofection and lipofection in adult fibroblasts. The cleavage,
transfection efficiency and cell viability of nucleofection group was found to be significantly (P ≤ 0.05) higher than lipofection group. Four MSTN edited colony were generated using nucleofection, out of which three colonies was found to be biallelic and one was monoallelic. Further, we compared the efficacy, embryonic developmental potential and subsequent pregnancy outcome of SCNT and zygotic electroporation. The blastocyst rate of electroporated group was found to be significantly (P ≤ 0.05) higher than SCNT group. However, the zygotic electroporation group resulted into two pregnancies which were confirmed to be MSTN edited. Since, the zygotic electroporation does not require complex micromanipulation techniques associated with SCNT, it has potential for facilitating the genetic modification in large livestock such as buffaloes. The present study lays the basis for inducing genetic alternation with practical or biological significance.