Cloning oocyte activation

The in vitro development of bovine cloned embryos was significantly improved by delayed activation with an electrical pulse for 4—6 h after fusion [18]. Other materials, including a calcium inducer (calcium iono-phore or ionomycin), a protein kinase inhibitor [6-dimethyl aminopurine (DMAP)] or a protein synthesis inhibitor (cyclohexa-mide or puromycin) were known oocyte activators. In cattle, incubation with cyclo-hexamide following exposure of the embryo to ionomycin or electrical pulses resulted in embryo development to term [19,... 

Definition of the background activity of microinjected oocytes is a clear prerequisite for any functional cloning procedure, as a positive signal needs to be distinguished from the endogenous transport activities. Key characteristics of the carrier, such as a dependency on sodium ions or a specific inhibitor are very helpful. In addition, unspecific binding of the radioactive transporter substrate to the oocyte membrane needs to be examined carefully. 

Corey JL, Davidson N, Lester HA, Brecha N, Quick MW (1994) Protein kinase C modulates the activity of a cloned gamma-aminobutyric acid transporter expressed in Xenopus oocytes via regulated subcellular redistribution of the transporter. J Biol Chem 269 14759-14767. 

DMAP, and reported the cleavage rate to be 90% and the blastocyst rate as 27%. Likewise, Cibelli et al. [8] used the same activation protocol to activate human reconstructed oocytes, but failed to obtain cloned human blastocysts. 

Since there is an absence of reports detailing the activation of human SCNT oocytes and the successful production of cloned blastocysts, it was necessary to identify several parameters, including the reprogramming time (the time between cell fusion and egg activation, returning the gene expression profile of the somatic cell to that needed for appropriate embryonic development) and activation methods. Based on results from animal SCNT oocytes and the parthenogenetic activation of human oocytes, we initially employed a porcine activation protocol (simultaneous fusion and activation with electrical pulse) which used hu-... 

Pig cloning will thus have a marked impact on tire optimization of meat production and xenohansplantation. To clone pigs from differentiated cells, tire nuclei of porcine Sus scrofa) fetal fibroblasts were injected into enucleated oocytes, and the development was induced by electro activation. The transfer of 110 cloned embryos to foiu sunogate mothers produced an apparently normal female piglet. The clonal provenance of the piglet was indicated by her coat color and confirmed by DNA microsatellite analysis. 

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