从大鼠不同生精细胞筛选精子发生过程中阶段特异表达基因

Screening the Stage-specific Expression Gene from Rat Different Germ Cells

目的 :从大鼠精原细胞、粗线期精母细胞和圆形精子细胞筛选精子发生过程中阶段特异表达基因。 方法 :用牛血清白蛋白梯度沉降法 (STAPUT法 ) ,分别从 9d龄和成年大鼠睾丸中 ,分离出处于减数分裂前、中、后的 3种生精细胞 ,即精原细胞、粗线期精母细胞和圆形精子细胞。运用RNA差异显示方法筛选差示cDNA。 结果 :本实验获得 19个cDNA差示片段 ,并克隆和测序出其中 6个cDNA序列 ,通过非重复序列 (nr)和EST序列同源比较 ,发现 5条cDNA分别与小鼠睾丸、附睾、乳腺、早期胚胎和大鼠卵巢基因高度同源 (88%～ 98% ) ,有一条未知新EST。结论 :差异显示技术是分离与精子发生有关基因的强有力工具。

Objectives: To screen the stage specific expression gene during rat spermatogenesis from rat spermatogonia, pachytene spermatocytes and round spermatids Methods: The highly enriched spermatogonia, pachytene spermatcoytes and round spermatids were isolated by sedimentation velocity at unit gravity, with use of 2%～4% BSA gradient in DMEM/F12 medium Then a differential mRNA display method was used for screening the stage specific expression gene Results : Nineteen differential cDNA segments were obtained Among them, 6 cDNAs were cloned and sequenced Using the BLAST Program to search GenBank databases, it revealed that ESTs of SG852, SC936, SC937,SG938 and SC940 were highly homologous (88%～98%) with the known ESTs derived from the mouse epididymis, mammary gland, testis, rat ovary and 16 mouse cell embryo respectively SC851 is unknown EST Conclusions: The differential display method is a powerful tool for screening the specific gene related to spermatogenesis