Reprogramming mechanism dissection and trophoblast replacement application in monkey somatic cell nuclear transfer.

Somatic cell nuclear transfer (SCNT) successfully clones cynomolgus monkeys, but the efficiency remains low due to a limited understanding of the reprogramming mechanism. Notably, no rhesus monkey has been cloned through SCNT so far. Our study conducts a comparative analysis of multi-omics datasets, comparing embryos resulting from intracytoplasmic sperm injection (ICSI) with those from SCNT. Our findings reveal a widespread decrease in DNA methylation and the loss of imprinting in maternally imprinted genes within SCNT monkey blastocysts. This loss of imprinting persists in SCNT embryos cultured in-vitro until E17 and in full-term SCNT placentas. Additionally, histological examination of SCNT placentas shows noticeable hyperplasia and calcification. To address these defects, we develop a trophoblast replacement method, ultimately leading to the successful cloning of a healthy male rhesus monkey. These discoveries provide valuable insights into the reprogramming mechanism of monkey SCNT and introduce a promising strategy for primate cloning.

Live birth of chimeric monkey with high contribution from embryonic stem cells.

A formal demonstration that mammalian pluripotent stem cells possess preimplantation embryonic cell-like (naive) pluripotency is the generation of chimeric animals through early embryo complementation with homologous cells. Whereas such naive pluripotency has been well demonstrated in rodents, poor chimerism has been achieved in other species including non-human primates due to the inability of the donor cells to match the developmental state of the host embryos. Here, we have systematically tested various culture conditions for establishing monkey naive embryonic stem cells and optimized the procedures for chimeric embryo culture. This approach generated an aborted fetus and a live chimeric monkey with high donor cell contribution. A stringent characterization pipeline demonstrated that donor cells efficiently (up to 90%) incorporated into various tissues (including the gonads and placenta) of the chimeric monkeys. Our results have major implications for the study of primate naive pluripotency and genetic engineering of non-human primates.

Nuclear transfer improves the developmental potential of embryos derived from cytoplasmic deficient oocytes.

Embryo development after fertilization is largely determined by the oocyte quality, which is in turn dependent on the competence of both the cytoplasm and nucleus. Here, to improve the efficiency of embryo development from developmentally incompetent oocytes, we performed spindle-chromosome complex transfer (ST) between in vitro matured (IVM) and in vivo matured (IVO) oocytes of the non-human primate rhesus monkey. We observed that the blastocyst rate of embryos derived from transferring the spindle-chromosome complex (SCC) of IVM oocytes into enucleated IVO oocytes was comparable with that of embryos derived from IVO oocytes. After transferring the reconstructed embryos into the uterus of surrogate mothers, two live rhesus monkeys were obtained, indicating that the nuclei of IVM oocytes support both the pre-and post-implantation embryo development of non-human primates.

Cloning of Monkeys by Somatic Cell Nuclear Transfer.

Somatic cell nuclear transfer (SCNT) is a promising method to establish genetically modified monkeys with identical genetic background as models in biomedical research. We have recently cloned monkeys by optimization of the SCNT protocols and inclusion of the epigenetic modulator. Here, we describe the protocol for generation of cloned monkeys by somatic cell nuclear transfer.

Developing ABEmax-NG with Precise Targeting and Expanded Editing Scope to Model Pathogenic Splice Site Mutations In Vivo.

RNA splicing is related to many human diseases; however, lack of efficient genetic approaches to modulate splicing has prevented us from dissecting their functions in human diseases. Recently developed base editors (BEs) offer a new strategy to modulate RNA splicing by converting conservative splice sites, but it is limited by the editing precision and scope. To overcome the limitations of currently available BE-based tools, we combined SpCas9-NG with ABEmax to generate a new BE, ABEmax-NG. We demonstrated that ABEmax-NG performed precise A⋅T to G⋅C conversion with an expanded scope, thus covering many more splicing sites. Taking advantage of this tool, we precisely achieved A⋅T to G⋅C conversion exactly at the splice sites. We further modeled pathogenic RNA splicing in vitro and in vivo. Taken together, we successfully generated a versatile tool suitable for precise and broad editing at the splice sites.

Cloning of a gene-edited macaque monkey by somatic cell nuclear transfer.

Cloning of macaque monkeys by somatic cell nucleus transfer (SCNT) allows the generation of monkeys with uniform genetic backgrounds that are useful for the development of non-human primate models of human diseases. Here, we report the feasibility of this approach by SCNT of fibroblasts from a macaque monkey (Macaca fascicularis), in which a core circadian transcription factor BMAL1 was knocked out by clustered regularly interspaced short palindromic repeat/Cas9 gene editing (see accompanying paper). Out of 325 SCNT embryos transferred into 65 surrogate monkeys, we cloned five macaque monkeys with BMAL1 mutations in both alleles without mosaicism, with nuclear genes identical to that of the fibroblast donor monkey. Further peripheral blood mRNA analysis confirmed the complete absence of the wild-type BMAL1 transcript. This study demonstrates that the SCNT approach could be used to generate cloned monkeys from fibroblasts of a young adult monkeys and paves the way for the development of macaque monkey disease models with uniform genetic backgrounds.