CRISPR/Cas9系统介导Slc6a6基因敲除大鼠的繁殖与鉴定

Reproduction and identification of Slc6a6 knockout rats mediated by the CRISPR/Cas9 system

目的利用CRISPR/Cas9技术构建牛磺酸转运体基因(solute carrier family 6 member 6,Slc6a6)敲除大鼠,繁殖并鉴定,为研究牛磺酸缺失对神经系统疾病的影响提供稳定的大鼠模型。方法针对Slc6a6基因第5外显子,设计向导RNA(single-guide RNA,sgRNA)介导Cas9核酸酶与靶点DNA特异性结合,并切割基因组DNA,被切割后的DNA进行重组修复,从而实现基因的敲除。通过基因型鉴定和测序分析检测新生大鼠基因型。利用Real-time PCR、Western blot技术和免疫组化等方法,分析大鼠脑组织的牛磺酸转运体(taurine transporter,TauT)的mRNA表达和蛋白表达,建立Slc6a6基因敲除大鼠模型。结果 F3代出现21只Slc6a6基因敲除纯合子(TauT^(-/-)),54只杂合子(TauT^(+/-)),27只阴性(TauT^(+/+)),F3代纯合率约为20.59%,基本符合孟德尔遗传定律。Slc6a6基因敲除纯合子大鼠脑组织mRNA水平基本不表达,TauT蛋白表达水平显著低于同窝阴性大鼠。结论本研究利用CRISPR/Cas9系统定向敲除Slc6a6基因,成功构建Slc6a6基因敲除大鼠模型。

Objective CRISPR/ Cas9 technology was used to construct a taurine transporter gene (solute carrier family 6 member 6, Slc6a6) knockout rat, which was used as a stable animal model to study the effects of taurine on neurological diseases. Methods For exon 5 of the Slc6a6 gene, a single-guide RNA (sgRNA) mediated the specific binding of Cas9 nuclease to the target DNA and cleaved the genomic DNA, which underwent recombinant repair to generate the gene knockout. Newborn rat genotypes were detected by genotyping and sequencing analysis. The mRNA expression and protein 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 F3 generation. The offspring showed a normal Mendelian ratio. No Slc6a6 mRNA was observed in brain tissues from Slc6a6 knockout homozygous rats and the expression level of TauT protein was significantly lower than that of littermate-negative rats. 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.