ABSTRACT: A group of adult somatic cell cloned mice were obtained by using cumulus cells as nuclei donor cells. To study the effect
of different nuclear transfer (NT) and activation methods on the development of mouse cloned embryos, embryos were reconstructed
using two traditional NT methods (electrofusion and direct injection) and four activation treatments (electric pulse, ethanol,
SrCl2 and electric pulse combined with SrCl2). The data showed that the efficiency of reconstruction using the direct injection method is significantly higher (90.7%)
than that of the electrofusion method (49.7%). Parthenogenetic embryos can develop to blastocyst stage with three activation
conditions, including ethanol, electric pulse and SrCl2; however, the rates of development to blastocyst after ethanol and electric pulse activation (52.4%, 54.2%) are significantly
lower than after SrCl2 activation (76.9%). Treatment of embryos for 6 h with 10 mmol/L SrCl2 was found to be the best condition for activation of parthenogenetic as well as reconstructed embryos. By contrast, reconstructed
embryos failed to develop to blastocyst stage after being activated by ethanol. The use of either injection or electrofusion
for embryo reconstruction affected the pre-implantation development. However, after transfer in pseudopregnant mice, cloned
mice were obtained from both methods.
Chinese Science Bulletin 04/2012; 52(2):209-214. · 1.32 Impact Factor
ABSTRACT: Therapeutic cloning, whereby embryonic stem cells (ESCs) are derived from patient-specific cloned blastocysts via somatic
cell nuclear transfer (SCNT), holds great promise for treating many human diseases using regenerative medicine. Teratoma formation
and germline transmission have been used to confirm the pluripotency of mouse stem cells, but human embryonic stem cells (hESCs)
have not been proven to be fully pluripotent owing to the ethical impossibility of testing for germ line transmission, which
would be the strongest evidence for full pluripotency. Therefore, formation of differentiated cells from the three somatic
germ layers within a teratoma is taken as the best indicator of pluripotency in hESC lines. The possibility that these lines
lack full multi-or pluripotency has not yet been evaluated. In this study, we established 16 mouse ESC lines, including 3
genetically defective nuclear transfer-ESC (ntESC) lines derived from SCNT blastocysts of infertile hermaphrodite F1 mice
and 13 ntESC lines derived from SCNT blastocysts of normal F1 mice. We found that the defective ntESCs expressed all in vitro markers of pluripotency and could form teratomas that included derivatives from all three germ layers, but could not be transmitted
via the germ line, in contrast with normal ntESCs. Our results indicate that teratoma formation assays with hESCs might be
an insufficient standard to assess full pluripotency, although they do define multipotency to some degree. More rigorous standards
are required to assess the safety of hESCs for therapeutic cloning.
Chinese Science Bulletin 04/2012; 53(23):3648-3655. · 1.32 Impact Factor
ABSTRACT: A simple monoculture system, combined with a chemically defined medium containing hepatocyte growth factor (HGF) and G5 supplement, was used to induce rhesus monkey embryonic stem cells (rESC) directly into neuroepithelial (NE) cells. Under these conditions, the generation of NE cells did not require the formation of embryoid bodies or co-culture with other cell types. The NE cells could further develop to generate neurons, astrocytes and oligodendrocytes. These results demonstrate a simple approach to obtain enriched and expandable populations of neural progenitors. Importantly, unlike other systems, the neural progenitors obtained using this approach may possess the potential to differentiate into various regional neural cells. Finally, the results suggest that the time-dependent shift in the differentiation potential of the rESC-derived neural progenitors in vitro reflects the developmental events that occur during neurogenesis in vivo. Thus, this system can be used to study the mechanisms of cell fate specification during non-human primate neurogenesis.
Reproductive biomedicine online 09/2009; 19(3):426-33. · 2.04 Impact Factor
ABSTRACT: Even though it generates healthy adults, nuclear transfer in mammals remains an inefficient process. Mainly attributed to abnormal reprograming of the donor chromatin, this inefficiency may also be caused at least partly by a specific effect of the cloning technique which has not yet been well investigated. There are two main procedures for transferring nuclei into enucleated oocytes: fusion and piezoelectric microinjection, the latter being used mostly in mice. We have, therefore, decided to compare the quality and the developmental ability, both in vivo and in vitro, of embryos reconstructed with electrofusion or piezoelectric injection. In addition, the effect of piezo setups of differing electric strengths was investigated. Along with the record of the rate of development, we compared the nuclear integrity in the blastomeres during the first cleavages as well as the morphological and cellular quality of the blastocysts. Our results show that the piezo-assisted micromanipulation can induce DNA damage in the reconstructed embryos, apoptosis, and reduced cell numbers in blastocysts as well as a lower rate of development to term. Even if piezo-driven injection facilitates a faster and more efficient rate of reconstruction, it should be used with precaution and with as low parameters as possible.
Reproduction (Cambridge, England) 06/2007; 133(5):947-54. · 3.09 Impact Factor
ABSTRACT: Cloning mammals by nuclear transfer is a powerful technique that is quickly advancing the development of genetically defined animal models. However, the overall efficiency of nuclear transfer is still very low and several hurdles remain before the power of this technique will be fully harnessed. Among these hurdles include an incomplete understanding of biologic processes that control epigenetic reprogramming of the donor genome following nuclear transfer. Incomplete epigenetic reprogramming is considered the major cause of the developmental failure of cloned embryos and is frequently associated with the disregulation of specific genes. At present, little is known about the developmental mechanism of reconstructed embryos. Therefore, screening strategies to design nuclear transfer protocols that will mimic the epigenetic remodeling occurring in normal embryos and identifying molecular parameters that can assess the developmental potential of pre-implantation embryos are becoming increasingly important. A crucial need at present is to understand the molecular events required for efficient reprogramming of donor genomes after nuclear transfer. This knowledge will help to identify the molecular basis of developmental defects seen in cloned embryos and provide methods for circumventing such problems associated with cloning the future application of this technology.
Reproductive Biology and Endocrinology 12/2003; 1:84. · 2.05 Impact Factor