Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo, Pirassununga, SP, Brazil.
Centre de Recherche en Reproduction et Fértilité, Faculty of Veterinary Medicine, University of Montreal, Saint-Hyacinthe, QC, Canada.
Methods Mol Biol. 2023;2647:225-244. doi: 10.1007/978-1-0716-3064-8_12.
Cloning by somatic cell Nuclear Transfer (SCNT) is a powerful technology capable of reprograming terminally differentiated cells to totipotency for generating whole animals or pluripotent stem cells for use in cell therapy, drug screening, and other biotechnological applications. However, the broad usage of SCNT remains limited due to its high cost and low efficiency in obtaining live and healthy offspring. In this chapter, we first briefly discuss the epigenetic constraints responsible for the low efficiency of SCNT and current attempts to overcome them. We then describe our bovine SCNT protocol for delivering live cloned calves and addressing basic questions about nuclear reprogramming. Other research groups can benefit from our basic protocol and build up on it to improve SCNT in the future. Strategies to correct or mitigate epigenetic errors (e.g., correcting imprinting loci, overexpression of demethylases, chromatin-modifying drugs) can integrate the protocol described here.
体细胞核移植（SCNT）克隆是一种强大的技术，能够将终末分化细胞重新编程为全能性，用于生成整个动物或多能干细胞，用于细胞治疗、药物筛选和其他生物技术应用。然而，由于 SCNT 获得活体和健康后代的成本高、效率低，其广泛应用仍然受到限制。在本章中，我们首先简要讨论导致 SCNT 效率低下的表观遗传限制以及目前克服这些限制的尝试。然后，我们描述了我们的牛 SCNT 方案，用于生产活体克隆牛，并解决核重编程的基本问题。其他研究小组可以从我们的基本方案中受益，并在此基础上进行改进，以提高未来的 SCNT 效率。可以整合本文描述的方案来纠正或减轻表观遗传错误的策略（例如，纠正印记位点、去甲基酶的过表达、染色质修饰药物）。
