Germ cell‐specific expression of GFP gene induced by chicken vasa homologue (Cvh) promoter in early chicken embryos

Abstract

In the present study, we cloned the promoter sequence of the chicken vasa homologue (Cvh) gene, and observed the induction of germ cell-specific gene expression regulated by Cvh promoter in vitro and in vivo. The 2,718-bp Cvh promoter sequence involved putative binding sites of transcription factors such as Oct-1, Nkx2-5, HNF-4 and v-Myb, and a putative CpG island. The reporter vectors, which expressed humanized recombinant green fluorescent protein (hrGFP) under the control of one of three lengths (827, 1,555 and 2,718 bp) of Cvh promoter, were transfected to the dispersed gonadal cells of 7-day incubated embryos in vitro. Every length of promoter-induced germ cell-specific hrGFP expression was verified by immunohistochemistry for germ cell-specific antigens, and the intensity of fluorescence was significantly higher in the transfected gonadal cells using 1,555- and 2,718-bp Cvh promoter compared with 827-bp promoter. These results suggested that the germ cell-specific and enhanced gene expression required at least a 1,555-bp sequence of the 5' flanking region of Cvh gene. In vivo transfection of the reporter vector with 1,555-bp Cvh promoter, which was performed by injecting liposome-DNA complex into the blood vessels of 2-day incubated embryos, also induced germ cell-specific hrGFP expression. The Cvh promoter could provide the basis for a methodology for the visualization, purification and genetical modification of germ cells, and might further contribute to our understanding of the development, proliferation, migration and differentiation of germ cells in the chicken.