Introduction and Expression of Recombinant Genes in Ascidian Embryos

In order to examine the expression of exogenous genes introduced into ascidian eggs, two recombinant plasmids pmiwZ and pHrMA4aCAT were microinjected into the cytoplasm of fertilized eggs of Ciona savignyi and Halocynthia roretzi, respectively. The plasmid pmiwZ contains the coding sequence of bacterial β-galactosidase gene (lac-Z) fused with animal gene promoters, while pHrMA4aCAT was constructed by fusing about 1.4-kb long 5' flanking region of H. roretzi muscle actin gene HrMA4a with bacterial chloramphenicol acetyltransferase gene (CAT). Injection of approximately 160 pl of 10 μg/ml pmiwZ DNA into Ciona eggs did not affect the embryogenesis, although introduction of the same volume of 30 μg/ml pmiwZ DNA resulted in abnormal development of injected eggs. When the expression of lac-Z was examined by histochemical detection of the enzyme activity, the expression was evident in the early tailbud embryos and later stage embryos, and larvae, irrespective of linear or circular form of the plasmid. The enzyme activity appeared in various cell-types including epidermis, nervous system, endoderm, mesenchyme, notochord, and muscle. In contrast, when pHrMA4aCAT was introduced into Halocynthia eggs and the appearance of CAT protein was examined later by the anti-CAT antibody, the CAT expression was restricted to muscle cells. These results indicate that the recombinant genes introduced into ascidian eggs could express during embryogenesis and that the 1.4-kb long 5' flanking region of HrMA4a contains regulatory sequences enough for the appropriate spatial and temporal expression of the gene.