Construction and identification of a mouse spermatocyte-derived cell line with a stable expression of PIAS-NY / 中华男科学杂志

<p><b>OBJECTIVE</b>To construct a lentiviral expression vector of the PIAS-NY gene, and establish a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.</p><p><b>METHODS</b>PIAS-NY was synthesized, amplified by PCR and cloned into the lentiviral vector expression plasmid pGC-FU. After digestion and sequencing, pGC-FU-PIAS-NY, pHelper 1.0 and pHelper 2.0 were co-transfected into 293T cells. Then the lentiviral particles were used to transfect the mouse spermatocyte-derived cells. The expression of the PIAS-NY protein was detected by Western blot.</p><p><b>RESULTS</b>We successfully constructed the lentiviral expression vector pGC-FU-PIAS-NY and established a mouse spermatocyte-derived cell line with a stable overexpression of PIAS-NY.</p><p><b>CONCLUSION</b>The construction of the lentiviral expression vector pGC-FU-PIAS-NY and the obtainment of stably transfected mouse spermatocyte-derived cells have paved the way for further studies on the roles of the PIAS-NY gene in spermatogenesis.</p>

Screening PIAS2-interacting proteins in the mouse using the yeast two-hybrid system / 中华男科学杂志

<p><b>OBJECTIVE</b>To screen and identify PIAS2-interacting proteins from the mouse spermatogonial cDNA library using the yeast two-hybrid system, and to investigate the action mechanism of PIAS2 in spermatogenesis.</p><p><b>METHODS</b>With pGBKT7-PIAS2 as a bait plasmid, the positive clones interacting with pGBKT7-PIAS2 were screened from the mouse spermatogonial cDNA library, the inserted fragments were sequenced and underwent bioinformatic analysis, and their interaction was verified using the yeast two-hybrid system.</p><p><b>RESULTS</b>Through screening, sequencing, homology analysis and yeast two-hybrid verification, we obtained 8 different candidate proteins interacting with PIAS2, including Cyfip2, Psmb3, Nmel, nischarin, Ints10, Nsun5, Gnb211 and Ndufaf3.</p><p><b>CONCLUSION</b>Eight different genes were successfully obtained using the yeast two-hybrid system, and their encoding proteins interacted with PIAS2, which might be related with male fertility regulation. Our findings have offered some new clues to the action mechanism of PIAS2 in spermatogenesis.</p>