The. Ggnbp1 And Ggn2 In Mouse Testis Expression And Interaction Studies

Primordial germ cells (PGC) are the precursors of the germ cells in the adult gonads. The germ cell deficient (gcd) mutation is a recessive, transgenic insertional mutation in which there is a drastic reduction of PGCs in the developing genital ridge, resulting in male and female infertility in the adult. The gcd phenotype is due to the deletion of a single gene, FancL, once called Pog. FANCL is involved in proliferation of PGCs in early embryo stage, and may play a role in the development of germ cells by interacting with a few testis-specific proteins in the adult testis. In a yeast two-hybrid screening FANCL was used as the bait to screen the adult testis cDNA libraries. A novel gene Ggn (gametogenetin), which encoded three putative proteins GGN1, GGN2 and GGN3 by alternative splicing, was identified. A novel gene Ggnbpl, indicating gematogenetin binding protein 1, was identified during the yeast two-hybrid screening of GGN1-interacting proteins. The function of these proteins remains unknown. Here, we have prepared GGN2, GGNBP1 and FANCL rabbit antiserum and immunoaffinity-purified specific antibodys. We used these antibodys to analyse the protein expression and interaction in vivo, and try to reveal their potential role in spermatogenesis.First, we cloned and expressed recombinant FANCL and GGNBP1. and obtained a 15 peptide of mouse GGN2 by chemical synthesis. Using them as antigens to immunize female rabbits, we have prepared rabbit antisera. We prepared antigen column by immoblization of purified antigens to HiTrap NHS-activated HP column, and immunoaffinity-purified the antibodies.Second, we prepared various mouse tissue lysates, and then applied the antibodies to these tissue lysates in Western blotting to check the protein expression in vivo. We have found that FANCL protein is widely expressed in mouse tissues. GGN2 and GGNBP1 are specifically expressed in the testis.Third, we checked the expression of Ggn and Ggnbpl in various tissues by Northern blotting. It was found that Ggn and Ggnbpl mRNAs were only transcriped in the adult testis but not in the adult ovary or other somatic tissues.Fourth, EGFP-GGN1 or EGFP-GGN2 was co-expressed with Myc-GGNBP1 by...