Tbx18-Cre基因敲入小鼠Cre基因保真性研究

Fidelity of Cre Gene in Tbx18-Cre Knock-in Mice

目的揭示Tbx18-Cre基因敲入小鼠cre基因表达的保真性。方法用实时荧光定量PCR（Real—timePCR）快速检测基因敲入小鼠外源基因Cre的相对拷贝数；JunctionPCR验证基因敲入小鼠整合位点Th。18-Cre的纯合性：利用Tbx18Cre。一纯合子小鼠模型验证Cre基因对下游基因的影响；利用Tbx18-Cre／Rosa26R．LacZ示踪模型，通过检测LacZ的表达，验证Cre基因的时空保真性。结果实时荧光定量PCR验证rkl8基因敲入小鼠外源基因Cre的相对拷贝数准确，JunctionPCR验证其整合位点特异。Thx18Cre-/-纯合子小鼠模型能特异性阻断转录因子Tbx18的表达，引起信号下游基因CX40、CX43、CX45的表达明砂升高。Tbx18-Cre／Rosa26R—LacZ示踪模型LacZ蛋白表达部位与Tbx18mRNA表达部位一致。结论7阢18一Cre基因敲入小鼠中Cre基因具有高度准确的时空保真性和特异性。

Objective To examine the fidelity of Cre gene in Tbx18-Cre knock-in mice. Methods Real-time fluorescence quantitative PCR was used to detect the relative copy number of Cre gene in knock-in mice and junction PCR to verify the homozygosity of Tbx18 in integration site. Tbx18 Cre-/-mice were employed to determine the effect of Cre gene on the downstream genes and the Tbx18-Cre/Rosa26R-LaeZ tracing model to verify the fidelity of Cre gene by detecting the expression of LacZ. Results Real-time PCR and junction PCR revealed that the relative copy num- ber of Cre gene was accurate and the integration site was specific in Tbxl 8 Cre knock-in mice. The expression of the transcription factor Tbx-18 could be specifically blocked in the Tbx18 Cre-/-mice model, which resulted in the increased expression of downstream genes CX40, CX43 and CX45. The expression site of LaeZ protein was sa26R-LacZ tracing model. Conclusion The fidelity. identical with that of Tbxl 8 mRNA In Tbxl8-Cre/RoCre gene in Tbx18-Cre knock-in mice possesses good