Pumilio家族基因诱导性敲除小鼠模型的构建
DOI:
作者:
作者单位:

南京医科大学

作者简介:

通讯作者:

中图分类号:

基金项目:

南京医科大学大型科学仪器开 放共享研究课题(ZC2021DY13)


The construction of Pumilio gene induced knockout mouse model
Author:
Affiliation:

Fund Project:

  • 摘要
  • |
  • 图/表
  • |
  • 访问统计
  • |
  • 参考文献
  • |
  • 相似文献
  • |
  • 引证文献
  • |
  • 资源附件
  • |
  • 文章评论
    摘要:

    目的:RNA结合蛋白PUMILIO家族参与神经、生殖等发育过程,其突变和表达异常可以导致神经性退化、发育阻滞及肿瘤的进程加速。为了探究出生后Pumilio家族的功能及其缺失对个体的影响,构建了Pumilio1 (Pum1)和Pumilio2 (Pum2)双基因诱导性敲除的小鼠模型。方法:通过小鼠交配实验得到R26-ERT2Cre/+;Pum1flox/flox;Pum2-/-小鼠,并将同窝雄鼠设为对照组和实验组。分别对实验组3周和成年小鼠注射他莫昔芬(Tamoxifen),在DNA、RNA和蛋白水平表征主要脏器中Pum1的敲除效果;并表征精子发生过程中的病理变化、细胞增殖及凋亡情况。结果:实验组3周和成年雄鼠的胸腺和睾丸组织中PUM1表达水平极低,敲除效率均高于50%,Pum1和Pum2双基因诱导性敲除小鼠模型构建成功。在注射他莫昔芬结束的第21天,3周雄鼠胸腺、脾脏和睾丸的重量明显下降,睾丸病理苏木精-伊红(HE,hematoxylin-eosin staining)染色实验显示精子形成障碍,TUNEL和BrdU染色后计数结果显示睾丸中生精细胞凋亡增加、增殖减慢。虽然,成年小鼠在注射他莫昔芬恢复后Pum1在胸腺、心、肝、睾丸中的表达仍有降低,但睾丸病理HE染色显示精子发生未见异常。结论:本文成功构建了诱导性PUM家族敲除模型,初步研究表明出生后3周Pumilio诱导性敲除后的小鼠睾丸重量显著下降,并出现明显的细胞凋亡增加和增殖减慢的缺陷;该模型的建立,首次展示PUM家族蛋白作为未来药物靶标的潜能,为深入研究PUM的功能及作为肿瘤治疗靶蛋白的研究奠定了理论基础。

    Abstract:

    Objective: To determine the roles of Pumilio family in the postnatal development, we established the Pumilio1/Pumilio2 (Pum1 and Pum2) inducible knockout mouse model. Methods: Pum1 flox/flox;Pum2 -/- mice were mated with R26-ERT2Cre/Cre mice to obtain R26-ERT2Cre /+;Pum1 flox/flox;Pum2 -/- mice. The experiment group (three weeks old mice and adult mice) was injected with tamoxifen, while the control group was injected with the same amount of peanut oil. The knockout efficiency of Pum1 at DNA, RNA and protein levels of multiple organs in mice was determined. The histology of the testis as well as cell proliferation and apoptosis of the testicular cells were examined. Results: The Pumilio1 RNA and protein of thymus and testis were knocked down significantly. The knock down efficiency of Pum1 varies from tissues to tissues but reaching a minimum of 50%. The mouse model of Pum1 and Pum2 gene induced knockout was successfully constructed. The weight of thymus, spleen and testis of male mice in the 3-week experiment group decreased significantly, supporting the critical role of PUM in postnatal organ growth.. The hematoxylin and eosin (HE), TUNEL and BrdU staining of testis pathology showed disrupted spermatogenesis, increased apoptosis and decreased proliferation of spermatogenic cells in 3 weeks experiment group testis. Although, the Pum1 of each organ in adult group was knocked down at different levels, the spermatogenesis of adult experimental group was not significantly affected. Conclusion: The inducible knockout of Pumilio family genes in the postnatal 3 weeks mice can lead to the weight loss of related organs. The inducible knock down of Pumilio gene in the testis may lead to testis weight reduction and disrupted spermatogenic process with increasing the apoptosis and decreasing proliferation of spermatogenic cells. Hence this inducible PUM knockout model provides a new tool to study the roles of PUM in postnatal and adult mice, and supports the future development of PUM as potential drug targets for diseases involving PUM-mediated translational control.

    参考文献
    相似文献
    引证文献
引用本文
分享
文章指标
  • 点击次数:
  • 下载次数:
  • HTML阅读次数:
  • 引用次数:
历史
  • 收稿日期:2022-09-27
  • 最后修改日期:2022-11-28
  • 录用日期:2023-08-09
  • 在线发布日期:
  • 出版日期: