Transgenic strategy for demonstrating nuclear reprogramming in the mouse.
Totipotency can be restored to the nuclei of somatic cells by reprogramming the nucleus via the technique of nuclear transfer. As genes expressed in somatic cells differ from those expressed in early embryos, a change in gene expression must accompany nuclear reprogramming. In this study, nuclear reprogramming of somatic cell nuclei following nuclear transfer (NT) was demonstrated by the reactivation of developmentally regulated lacZ reporter genes. NT embryos were generated by direct injection of adult cumulus cell nuclei into mature mouse oocytes from which maternal chromosomes were subsequently removed. Cumulus cells were collected from transgenic mice which show developmentally regulated lacZ reporter gene expression as a result of integration and functional coupling of reporter gene expression with the endogenous Oct4 or COB54 genes. As both genes are transcriptionally silent in somatic cells but are expressed during early embryonic development, reactivation of reporter gene expression in cumulus cell-derived NT embryos was assessed as a means of monitoring nuclear reprogramming. The pattern of X-gal staining observed in individual NT embryos derived from both transgenic lines revealed that coordination of reactivation appeared to be specific for each gene, and that the timing of expression was consistent with that seen in control non-manipulated transgenic embryos. However, the staining in some NT embryos appeared to be diminished or absent. This reduction in staining may indicate a failure to reprogram gene expression in these embryos. Similar transgenic strategies employing lacZ vital stains, or alter-native reporters such as GFP, may facilitate real-time monitoring of reprogramming and a potential selection strategy to increase cloning efficiency through the identification and selection of reprogrammed, preimplantation stage NT embryos prior to embryo transfer.
Centre for Early Human Development, Monash Institute of Reproduction and Development, Monash Universitym Clayton, Victoria, Australia.
Megan
M
Munsie
Carmel
C
O'brien
Peter
P
Mountford
D000818:Animals; D002467:Cell Nucleus; D046508:Culture Techniques; D005314:Embryonic and Fetal Development; D005260:Female; D017930:Genes, Reporter; D007763:Lac Operon; D051379:Mice; D008810:Mice, Inbred C57BL; D008811:Mice, Inbred DBA; D008822:Mice, Transgenic / Q000235:genetics*