Nuclear transfer: the importance of donor and recipient cells for nuclear reprogramming and cloning efficiency in mammals
Wells, David N
Background: Fourteen years after the birth of Dolly, it is still remarkable that nuclear transfer (NT) using differentiated cells can produce physiologically normal animals. But the process is highly inefficient and prone to epigenetic errors leading to faulty reprogramming of the donor genome following NT. At present in cattle, only 9% of the embryos transferred to recipient females typically result in cloned adults, with continual losses throughout development. Importantly, it appears that any epigenetic errors in surviving clones are corrected during gametogenesis and not transmitted to sexually-derived offspring. Moreover, the composition of milk and meat from cloned livestock is normal. This provides confidence for the use of NT in animal breeding to disseminate genetic gain. In addition, NT has advantages in the production of transgenic farm animals, especially for biomedical applications. Review: The importance of donor and recipient cells on the efficiency of nuclear reprogramming and cloning efficiency is discussed. The reprogrammability of donor cells is influenced by differentiation status, (epi)genotype and cell cycle state. Embryonic blastomeres, and some embryonic stem cell lines, are more easily reprogrammed and result in higher cloning efficiency than more differentiated cells. However, somatic stem cells are not consistently better nuclear donors than differentiated isogenic progeny. Genotype influences NT success, with both hybrid donor and recipient cells proving advantageous. Optimal in vitro conditions are critical to maintain genetic and epigenetic stability of cultured cells. Appropriate epigenetic markers may be useful in identifying more clonable cell lines. Amongst those cell cycle combinations and protocols compatible with better maintaining normal ploidy after NT, the use of G0 donor cells with recipient oocytes results in twice the cloning efficiency to adulthood compared to the use of G1 donors. For somatic cell NT, mature metaphase II oocytes are commonly used as recipients but it is apparent that immature oocytes, zygotes and even totipotent blastomeres also have significant reprogramming ability. Crucial reprogramming factors appear sequestered in the nucleus. Hence, meiotic or mitotic stages, when these factors are redistributed in the cytoplasm, are required to enable successful somatic cell cloning. The benefits of zygotic cytoplasm may arise from sperm-mediated activation and introduction of additional RNAs. Zygotic and early embryonic factors may aid development following serial NT strategies. These transfer the nucleus firstly into a mature oocyte for initial reprogramming and then into a zygotic or embryonic cytoplasm in a second NT step. Increasing the cytoplasmic volume of the recipient, achievable with zona-free NT methods, provides more reprogramming factors to promote embryogenesis. A promising approach involves chromatin therapy to remove the epigenetic constraints on the donor chromatin and has substantially increased cloning efficiency in the mouse. Conclusion: Given the continual problems with somatic cell NT and advances in genomic selection of livestock genotypes along with rapid advances in induced pluripotency of somatic cells, a return to embryonic cloning and renewed interest in the establishment of embryonic stem cells in farm animal species is occurring. The discovery of reprogramming factors to complement those present in the oocyte that reliably result in full reprogramming will advance somatic cell NT. The improved viability and health of animals produced following NT will contribute towards greater market acceptance and use of cloned (and transgenic) livestock in (ph)farming systems.
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