Isolation of sexual differentially expressed genes in the chicken embryonic gonads during early sexual-differentiation period by using suppression subtractive hybridization

Isolation of sexual differentially expressed genes in chicken embryonic gonads during early sexual-differentiation period could provide basic information for studies on the genetic mechanism of avian sex determination and sex differentiation. In present study, total RNAs from female and male chicken embryonic gonads at days 3.5-6 post incubation were used to construct, respectively, forward and reverse subtracted cDNA libraries by using the suppression subtractive hybridization, and 39 positive differentially expressed cDNA clones were isolated from the libraries by dot blotting. The subtractive efficiency of 2~5 folds was obtained in the two libraries by using the housekeeping gene encoding glyceraldehyde-3-phosphate dehydrogenase(GAPDH) as the reference. PCR analysis showed that most of the differentially expressed clones contained inserts of 250-750 bp. Dot blot analysis of 252 and 168 cDNA clones respectively from female and male cDNA libraries identified 39 differentially expressed clones. The sequence analysis of these 39 clones revealed that they represented 18 known genes and 11 putative genes respectively localized on different chromosomes. Based on the function of the homologous genes in mammals, 18 known genes identified here were predicted to involve in multiple biological processes. The expression of 10 genes, respectively, five from female subtracted library and five from male’s, was further analyzed by semi-quantitative RT-PCR and results showed that nine of ten genes had the significant different expression between females and males with the exception of one gene from female cDNA library. The obtained sexual differentially expressed genes would provide the bases for further research on the regulation of gene expression during chicken gonadal development.