睾丸间质细胞Prl3c1过表达转基因小鼠建立与生物学功能初步研究

目的 建立睾丸prolactin family 3,subfamily c,member 1(Prl3c1)过表达转基因(TG)小鼠,并对其功能进行初步研究.方法 设计引物扩增insulin like 3(Insl3)、Prl3c1、Insl3PA片段,通过融合PCR,形成Insl3-Flag-Prl3c1-Insl3 PA片段,构建pPrl3c1转基因载体,测序验证.通过受精卵原核注射,建立TG小鼠.免疫荧光、Western blot方法鉴定转基因表达情况.采用ELISA方法检测3月龄TG小鼠与野生型对照(WT)小鼠睾酮基础水平;并测定5 U/10 g人绒毛膜促性腺激素(human chorionicgonadotropin,HCG)刺激后,TG与WT小鼠睾酮水平(n=6).结果 免疫荧光与Western blot检测结果表明转基因片段表达于TG小鼠睾丸间质细胞.TG小鼠睾丸形态学未见明显异常.此外,TG小鼠基础睾酮水平与WT小鼠差异无统计学意义[(2.61 ±0.34) ng/mL vs (2.51 ±0.35) ng/mL,P＞0.05],但在HCG刺激后,TG小鼠睾酮水平低于WT小鼠[(5.35±0.83) ng/mL vs(7.56±1.17) ng/mL,P＜0.01].Western blot检测结果表明Prl3c1过表达减弱HCG刺激的STAR表达(P＜0.01).结论 成功建立了Prl3c1 TG小鼠,体内功能研究显示Prl3c1参与睾酮产生.

Establishment and biological functions of a transgenic mouse with Prl3c1 overexpression in testicular Leydig cells

Objective To generate prolactin family 3,subfamily c,member 1 (Prl3c1) transgenic (TG) mice and investigate its function during male reproduction.Methods The transgenic construct (Insl3-Flag-Prl3c1-Insl3PA) was generated by in-fusion PCR with designed primers to amplify the fragments of insulin like 3 (Insl3),Prl3cl and Insl3PA,respectively.After being sequenced,the pPrl3c1 transgenic constructs were microinjected into the pronuclei of fertilized oocytes from mice.The founder (F0) TG mice were screened by PCR.Western blotting and immunofluorescence assay were performed to detect the expression of transgenic construct in the testis.Additionally,basic and 5 U/10 g human chorionic gonadotropin (HCG)-stimulated testosterone levels of the TG and wild type (WT) mice (6 animals in each group) were measured using enzyme-linked immunosorbent assay (ELISA).Results Western blotting and immunofluorescence staining showed that the Prl3c1 transgenic construct was expressed in the Leydig cells.The testes of TG mice were morphologically normal in appearance.There was no difference in serum testosterone levels between TG mice and WT mice (2.61 ± 0.34 vs 2.51 ± 0.35 ng/mL,P ＞ 0.05).However,HCG stimulation caused testosterone production lowered in TG mice compared with WT mice (5.35 ±0.83 vs 7.56 ± 1.17 ng/mL,P ＜0.01).Further,Western blotting indicated that Prl3c1 over-expression caused a decrease in HCG-stimulated STAR expression in TG mice (P ＜ 0.01).Conclusion Prl3c1 TG mice are successfully established.Additionally,Prl3c1 may be involved in testosterone production.