Gibel carp germ cell marker Vasa: cDNA cloning and its antibody production (English)

摘要

The basis for animal reproduction and reproductive biotechnology is germ cells that are segregated from the somatic lineage early in embryonic development and produce sperm and eggs for germline transmission between generations. To provide a germ cell marker, we cloned and characterized Cagvasa, a vasa homolog from the gibel carp (Carassius auratus gibelio). The Cagvasa cDNA is 2 771 nt (nucleotide) and encodes a 701-aa(amino acid) protein (CagVasa) that possesses 14 RGG-repeats and all eight conserved motifs of Vasa proteins. CagVasa shows sequence identities of 95%, 89%, 61%–66% and 50% to Vasa proteins from the common carp, zebrafish, tetrapods and Drosophila, respectively. RNA in situ hybridization on ovarian sections showed that Cagvasa was absent in any gonadal somatic cells but dynamically expressed throughout oogenesis: the signal is weak in oogonia, peaks in vitellogenic oocytes and deceases in maturing oocytes. To develop a tool for analyzing fish germ cells, we generated aVasa, a rabbit antibody against a recombinant protein of 310-aa N-terminal CagVasa. Western blot analyses showed that aVasa detected a 75 kD protein exclusively in the gibel carp gonads and isolated oocytes. To confirm the germ cell-specifity of aVasa, fluorescent immunostaining was performed on gonadal sections. This antibody stains exclusively germ cells throughout oogenesis and early stages of spermatogenesis. We conclude that vasa encodes a Vasa ortholog that services a first germ cell marker in the gibel carp, and that aVasa provides a highly sensitive and powerful tool for the analysis of germ cell specification and differentiation in the gibel carp[Acta Zoologica Sinica 51(4):732–742, 2005].