Somatic Cell Nuclear Transfer Reprogramming: Mechanisms and Applications

Abstract

Successful cloning of monkeys, the first non-human primate species, by somatic cell nuclear transfer (SCNT) attracted worldwide attention earlier this year. Remarkably, it has taken more than 20 years since the cloning of Dolly the sheep in 1997 to achieve this feat. This success was largely due to recent understanding of epigenetic barriers that impede SCNT-mediated reprogramming and the establishment of key methods to overcome these barriers, which also allowed efficient derivation of human pluripotent stem cells for cell therapy. Here, we summarize recent advances in SCNT technology and its potential applications for both reproductive and therapeutic cloning.

Keywords: epigenetic reprogramming; human disease model generation; iPSC; reproductive cloning; somatic cell nuclear transfer; therapeutic cloning.

Figure 1.

Diagram showing the major steps of reproductive and therapeutic cloning compared to natural fertilization. (Top) Natural fertilization; metaphase II (MII stage) oocytes are activated by fertilized sperm and form paternal and maternal pronuclei (PN), and continue preimplantation cleavages until they reach the blastocyst stage. (Middle) Reproductive cloning; donor somatic cell nuclei introduced into the enucleated oocytes quickly undergoes nuclear membrane break down (NMBD) to form metaphase chromosome in a process called premature chromosome condensation (PCC). The reconstructed SCNT oocytes are artificially activated to initiate developmental program to form blastocysts. (Bottom) Therapeutic cloning; patient derived somatic cells are introduced to enucleated oocytes similar to reproductive cloning. Pluripotent embryonic stem cells can be derived from the blastocysts of nuclear transferred embryos (ntESCs), and the causative mutation can be corrected in vitro if desired.

Figure 2.

Molecular mechanisms of SCNT reprogramming and its associated barriers in reproductive cloning. (A) Diagram showing the cellular and molecular events taking place in the SCNT reprograming process. PPN; pseudo pronuclei. ZGA; zygotic genome activation. DHSs; DNase I hypersensitive sites. TF; transcription factor. CGI; CpG island. (B) Diagram illustration of the various epigenetic barriers in reproductive cloning. XCI; X chromosome inactivation. LOI; loss-of-imprint.

Figure 3.

Application of SCNT technology in human disease model generation. CRISPR/Cas9-mediated genome editing coupled with improved SCNT cloning enables rapid generation of human disease animal models. CRISPR/Cas9-mediated genome editing can induce deletion to produce gene knockout (KO) alleles, or insertion to produce knockin (KI) alleles, or replacement of specific pathogenic mutation via HDR.