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    利用TALEN系统对兔进行Tiki1基因敲除

    Gene knockout of Tiki1 gene in rabbit by TALEN system

      摘要
    • 摘要:
      目的  利用转录激活样效应因子核酸酶(TALEN)系统获得Tiki1基因敲除的兔模型，为研究Tiki1对动物早期发育作用机理提供兔源的动物模型。
      方法  基于TALEN系统设计了靶向兔Tiki1基因的打靶载体，分别将10和50 ng/μL的Tiki1-TALEN mRNA注射到原核期的家兔受精卵胞质中，然后收集发育至囊胚期的胚胎，鉴定囊胚率和基因修饰效率，通过测序检验囊胚的基因突变。为了进一步获得Tiki1基因敲除兔，后续将50 ng/μL的Tiki1-TALEN mRNA注射到17个原核期的家兔受精卵胞质中，将受精卵分别移植到2只受体兔体内。
      结果  注射50 ng/μL Tiki1-TALEN mRNA试验组的囊胚率（64%）与注射10 ng/μL Tiki1-TALEN mRNA试验组的囊胚率（57%）差异不大，但注射50 ng/μL Tiki1-TALEN mRNA试验组的囊胚基因修饰效率（100%）显著高于注射10 ng/μL Tiki1-TALEN mRNA试验组的囊胚基因修饰效率（14.3%）。测序结果表明，Tiki1基因的突变范围从 1到28 bp缺失不等。经过胚胎移植共生出3只仔兔，其中有2只兔子能检测到基因突变。
      结论  所建立的TALEN技术体系可以对家兔Tiki1基因进行高效的敲除。

       

      Abstract:
      Objective  To obtain a rabbit model of Tiki1 gene knockout by the transcription activator-like effector nuclease (TALEN) system, and provide a rabbit model for investigating the mechanism of Tiki1 gene on early development of animals.
      Method  Using TALEN system, the target vector of rabbit Tiki1 gene was constructed and then 10 or 50 ng/μL Tiki1-TALEN mRNA was injected into the cytoplasm of fertilized eggs at pronuclear stage. Embryos developed to blastocyst stage were collected. The blastocyst rate and gene modification efficiency were investigated. To further obtain Tiki1 knockout rabbits, 50 ng/μL Tiki1-TALEN mRNA was subsequently injected into the cytoplasm of 17 prokaryotic fertilized eggs of rabbit, and then the fertilized eggs were transplanted into two recipient rabbits.
      Result  The blastocyst rate of the treatment group injected with 50 ng/μL Tiki1-TALEN mRNA（64%) was almost the same as that of the group injected with 10 ng/μL Tiki1-TALEN mRNA(57%), while the blastocyst gene modification efficiency of the group injected with 50 ng/μL Tiki1-TALEN mRNA(100%) was significantly higher than that of the group injected with 10 ng/μL Tiki1-TALEN mRNA (14.3%). The sequencing results showed that mutations of Tiki1 gene ranged from the deletion of 1 bp to 28 bp. A total of three rabbits were born after embryo transfer, and two of them were detected with genetic mutations.
      Conclusion  The TALEN technology system established in this study could effectively knock out Tiki1 gene in rabbit.

       

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