Transgenesis of newt with exogenous gene expression facilitated by satellite 2 repeats

Abstract

Genetiç transduction of newt was undertaken in search of conditions allowing high exogenous gene expression in stages from late embryo to larva when regeneration experiments, which are advantageous for this group of animals, are possible. DNA of a β-galactosidase-encoding gene miwZ was injected in the cytoplasm of fertilized eggs in different molecular forms, and its fate and gene expression were studied in individual embryos. When DNA was injected in a linear form, DNA concatemerized quickly and the majority of the exogenous sequences were localized in the nuclear fraction from early gastrula onwards. By contrast, when circular DNA was injected, the exogenous DNA sequence largely remained in the cytoplasmic fraction, and the entry of the sequence into the nuclear fraction was delayed to mid-gastrula stage. Reflecting these differences, β-galactosidase expression in an embryo occurred earlier and more efficiently by linear DNA injection. Whole mount staining for β-galactosidase activity in tail bud embryos showed only spotty staining with circular DNA injection while spreading staining was also observed with linear DNA, suggesting difference in clone sizes of expressing cells depending on the DNA form. Expression of β-galactosidase, however, became low in the hatching stage. To augment and stabilize β-galactosidase expression to this stage, repeats of satellite 2 sequence, which is a transcriptionally active middle repetitive sequence highly conserved among urodeles, were added to the DNA in a way to bracket the miwZ gene. Injection of this DNA in linear form resulted in one order of magnitude higher β-galactosidase expression compared to without satellite 2 in the neurula embryos, and in high and persistent expression in the hatching larvae, without significant increase in the copy number of the exogenous DNA. Satellite 2 sequence probably provided a genetic environment favorable for transcription of a gene.