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Home > Archive>Volume 29, Issue 5, 2019 >44-50. DOI:10.3969/j. issn. 1671 -7856. 2019. 05. 007
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Reproduction and identification of Slc6a6 knockout rats mediated by the CRISPR / Cas9 system
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(1. Tianjin Medical University, Tianjin 300070, China. 2. Tianjin Huanhu Hospital, Neurosurgery Institute of Tianjin,Tianjin Key Laboratory of Cerebrovascular and Neurodegenerative Diseases, Tianjin 300350)

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R-33

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    Abstract:

    Objective CRISPR/ Cas9 technology was used to construct a taurine transporter gene (solute carrierfamily 6 member 6, Slc6a6) knockout rat, which was used as a stable animal model to study the effects of taurine onneurological diseases. Methods For exon 5 of the Slc6a6 gene, a single-guide RNA (sgRNA) mediated the specificbinding of Cas9 nuclease to the target DNA and cleaved the genomic DNA, which underwent recombinant repair to generatethe gene knockout. Newborn rat genotypes were detected by genotyping and sequencing analysis. The mRNA expression andprotein expression of TauT in rat brain tissues were analyzed by real-time PCR, Western blot and immunohistochemistry.The homozygous Slc6a6 knockout rat was screened out. Results The F3 progeny had 21 Slc6a6 knockout homozygotes(TauT-/ - ), 54 heterozygous (TauT+/ - ) and 27 wild-types (TauT+/ + ). The homozygosity rate was about 20. 59% in the F3generation. The offspring showed a normal Mendelian ratio. No Slc6a6 mRNA was observed in brain tissues from Slc6a6knockout homozygous rats and the expression level of TauT protein was significantly lower than that of littermate-negativerats. Conclusions In this study, the CRISPR/ Cas9 system is used to knockout the exon of the Slc6a6 gene, and the Slc6a6 knockout rat model is successfully constructed.

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History
  • Received:January 17,2019
  • Online: June 05,2019
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