Nuclear transplantation from stably transfected cultured cells of Xenopus.
By nuclear transplantation we have generated embryos from enucleated Xenopus eggs and nuclei of stably transfected Xenopus cell lines. We have devised a novel method of transplantation in which cell permeabilization is controlled by a temperature effect on streptolysin O-treated cells. This method is easier and quicker to operate than the conventional cell rupture technique. Single nuclei from cell lines transfected with the lacZ reporter gene were transplanted to Xenopus eggs in which the egg nuclei were destroyed by UV irradiation. We show that the lacZ transgene is transmitted from donor cells to nuclear transplant embryos. Expression of the lacZ transgene has been controlled by the elongation factor 1-alpha promoter (Krieg et al., Dev. Biol. 133: 93-100, 1989). In the nuclear transplant embryos, beta-galactosidase transcripts are expressed at the expected time of development, that is after the mid-blastula transition. In addition, we show that early embryo-specific genes, not expressed in cultured cells, are normally activated in nuclear transplant embryos. Therefore, expression of these genes can be used to monitor the effects of transfected test genes. Although most of the nuclear transplant embryos do not develop beyond the gastrula stage, explants of equatorial tissue from these embryos can undergo differentiation characterized by the expression of muscle and notochord markers. The use of nuclear transplantation, as described here, provides a means of avoiding the mosaic expression of DNA or mRNA injected into Xenopus eggs.
Chan
A P
AP
Wellcome/CRC Institute, University of Cambridge, England.
Gurdon
J B
JB
D000818:Animals; D030801:Animals, Genetically Modified / Q000196:embryology*; D015415:Biomarkers / Q000032:analysis; D002454:Cell Differentiation / Q000235:genetics; D002478:Cells, Cultured / Q000502:physiology; D046508:Culture Techniques; D018507:Gene Expression Regulation, Developmental; D017781:Genes, Immediate-Early / Q000235:genetics; D017874:Immediate-Early Proteins / Q000096:biosynthesis; D007763:Lac Operon / Q000235:genetics; D008648:Mesoderm / Q000378:metabolism; D053652:Nuclear Transfer Techniques; D013301:Streptolysins / Q000494:pharmacology; D014162:Transfection / Q000235:genetics; D019076:Transgenes / Q000235:genetics; D014180:Transplantation / Q000379:methods*; D014981:Xenopus / Q000196:embryology*
D015415:Biomarkers; D017874:Immediate-Early Proteins; D013301:Streptolysins