Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene withXenopus cardiacα-actin promoter after injection intoXenopus fertilized eggs

Abstract

Circular and linearized plasmid DNA which contained bacterial chloramphenicol acetyltransferase (CAT) gene connected toXenopus cardiacα-actin promoter was injected intoXenopus fertilized eggs to study their expression in the course of early embryonic development. While circular DNA was slightly replicated and expressed only after embryos reached neurula stage, linearized DNA formed a large amount of concatemers, and was expressed as early as at blastula stage, or about 14 hr earlier than the time of circular DNA expression. Similarly earlier expression of linearized DNA occurred slightly more strongly when the DNA was injected into presumptive dorsal than in ventral blastomeres at 4-cell stage, and the expression was not affected when embryos were dissociated at blastula stage and their cells were cultured under reaggregating or nonreaggregating conditions. These results show that although circular actin-CAT fusion gene is expressed during development according to endogenous temporal control, the expression of linearized DNA deviates from such developmental control even though it contains intact promoter ofα-actin gene. It is then recommended that study of the control of the expression of exogenously-introduced DNA inXenopus fertilized eggs should be carried out with circular but not linearized plasmids.

Keywords: CAT enzyme assay; DNA microinjection; Xenopus eggs; linearized DNA; α-actin gene promoter.