Construction of a yeast two-hybrid cDNA library from the human testis / 中华男科学杂志

ABSTRACT

<p><b>OBJECTIVE</b>To construct a human testis cDNA library for yeast two-hybrid screening.</p><p><b>METHODS</b>Human normal testis mRNA was purified from total RNA, and ds cDNA was synthesized and amplified using primers SMART III and CDS III oligo (dT) as the base of recombination. The purified PCR products and linearized plasmid pGADT7-Rec were co-transformed into the competent yeast Y187 and recombined by yeast homologous recombinase in the yeast cells to form an active cyclic plasmid. All the clones growing on the SD/-Leu plates were harvested to constitute a human testis cDNA library.</p><p><b>RESULTS</b>We constructed a human testis cDNA library with high multiplication and adequate capacity, from which 2.0 x 10(6) recombinants were obtained. The amplified PCR fragments were between 0.3 kb and 4.0 kb in length.</p><p><b>CONCLUSION</b>The yeast two-hybrid cDNA library of human testis was successfully constructed by the Clontech SMART method, which has prepared a ground for further studies on the molecular mechanism of spermatogenesis.</p>