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  • ISSN 1006-3080
  • CN 31-1691/TQ

Kap147/Kpn杂合启动子引导Cre重组酶在转基因小鼠睾丸组织中特异性表达

刘飞 孟军 范立强

刘飞, 孟军, 范立强. Kap147/Kpn杂合启动子引导Cre重组酶在转基因小鼠睾丸组织中特异性表达[J]. 华东理工大学学报(自然科学版), 2019, 45(6): 919-927. doi: 10.14135/j.cnki.1006-3080.20180830001
引用本文: 刘飞, 孟军, 范立强. Kap147/Kpn杂合启动子引导Cre重组酶在转基因小鼠睾丸组织中特异性表达[J]. 华东理工大学学报(自然科学版), 2019, 45(6): 919-927. doi: 10.14135/j.cnki.1006-3080.20180830001
LIU Fei, MENG Jun, FAN Liqiang. A Testis-Specific Expression of Cre Recombinase in Transgenic Mice Testis by Kap147/Kpn Hybrid Promoter[J]. Journal of East China University of Science and Technology, 2019, 45(6): 919-927. doi: 10.14135/j.cnki.1006-3080.20180830001
Citation: LIU Fei, MENG Jun, FAN Liqiang. A Testis-Specific Expression of Cre Recombinase in Transgenic Mice Testis by Kap147/Kpn Hybrid Promoter[J]. Journal of East China University of Science and Technology, 2019, 45(6): 919-927. doi: 10.14135/j.cnki.1006-3080.20180830001

Kap147/Kpn杂合启动子引导Cre重组酶在转基因小鼠睾丸组织中特异性表达

doi: 10.14135/j.cnki.1006-3080.20180830001
基金项目: 国家自然科学基金项目(81071252)
详细信息
    作者简介:

    刘飞:刘 飞(1989-),男,山西吕梁人,硕士生,研究方向为基因表达和调控。E-mail:feilenw@126.com

    通讯作者:

    范立强,E-mail:fanglq@ecust.edu.cn

  • 中图分类号: Q291

A Testis-Specific Expression of Cre Recombinase in Transgenic Mice Testis by Kap147/Kpn Hybrid Promoter

  • 摘要: 为了验证杂合启动子Kap147/Kpn的体内、外功能,构建了Kap147/Kpn引导的Cre重组酶转基因鼠。体外细胞转染和激素诱导证实Kap147/Kpn启动子具有细胞特异性,且受雄激素诱导。通过RT-PCR(Reverse Transcription-Polymerase Chain Reaction)和荧光定量PCR(Polymerase Chain Reaction)证实Kap147/Kpn启动子引导Cre重组酶在转基因鼠(Cre-F0-2)睾丸组织中特异表达。荧光定量PCR检测发现,Cre mRNA在转基因鼠睾丸中的表达量与小鼠年龄相关,经15 mg/(kg·d)二氢睾酮(DHT)处理后睾丸中Cre mRNA表达量增加1.7倍,经100 mg/(kg·d)醋酸环丙孕酮(CAP)处理后Cre mRNA表达量降低约50%,证实Kap147/Kpn启动子在转基因鼠体内受雄激素调控。荧光组化显示,转基因鼠Cre-F0-2与双荧光Cre重组酶报告鼠杂交后,能成功介导子代鼠睾丸组织特异性的基因敲除。杂合启动子Kap147/Kpn引导的Cre重组酶转基因鼠(Cre-F0-2)具有雄激素调节性和睾丸靶向性,是一个潜在的研究睾丸基因功能的有力工具。

     

  • 图  1  基因结构和基因重组原理示意图

    Figure  1.  Scheme of structure and recombination of gene

    图  2  不同Kap启动子片段引导Cre重组酶在OK细胞中的表达活性和雄激素诱导性分析

    Figure  2.  Cre recombinase expression and Androgen regulated analysis driven by Kap promoter in OK cells

    图  3  Kap147/Kpn启动子引导Cre重组酶的细胞特异性表达    

    Figure  3.  Cell-specific Cre recombinase expression driven by Kap147/Kpn hybrid promoter in cultured cells

    图  4  Cre基因和Kap基因在Cre-F0-2转基因小鼠不同组织中的表达

    Figure  4.  Transgenic Cre and endogenerous Kap gene expression in the body tissue of transgene mice Cre-F0-2

    Data represent “mean±SD” (n=3); Symbols: Plus sign means normal RT-PCR reaction, Minus sign means RT-PCR reaction without RT enzyme

    图  5  Cre基因在睾丸组织中的表达受雄激素的调控

    Figure  5.  Androgen regulated expression of Cre gene in the testis

    Data represent “mean±SD” (n=3), *P≤0.05; Plus sign means normal RT-PCR reaction; Minus sign means RT-PCR reaction without RT enzyme; H means RT-PCR reaction without RNA template; M means DNA marker; G means RT-PCR reaction with standard positive plasmids

    图  6  Cre重组酶在双荧光报告鼠体内介导基因重组

    Figure  6.  Gene recombination mediated by cre recombinase in double fluorescent transgenic mice

    mT—Membrane-targeted tandem dimer tomato; mG—Membrane-targeted green fluorescent protein

    表  1  RT-PCR引物序列

    Table  1.   Primer sequence of RT-PCR

    PrimersSequence
    Actin-F5’-CCATCTCCTGCTCGAAGTCT-3’
    Actin-R5’-CCATCTACGAGGGCTATGCT-3’
    Cre-F5’-TGTTTCACTGGTTATGCGGC-3’
    Cre-R5’-AGTCATCCTTAGCGCCGTAA-3’
    Kap-F5’-ACTGTGGCTTTCCCCCTGTC-3’
    Kap-R5’-CTTCCTCGTTCTTTCTTCTTTG-3’
    下载: 导出CSV
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出版历程
  • 收稿日期:  2018-08-30
  • 网络出版日期:  2019-10-09
  • 刊出日期:  2019-12-01

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