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目的 克隆大鼠代谢型谷氨酸受体 1亚型 (mGluR1 )基因特异片段 ,制备cRNA探针。方法 从Wistar大鼠小脑中提取总RNA ,以RT PCR方法得到预期的 599bp条带 ,将这一片段克隆到pGEM Teasy载体上 ,经酶切鉴定正确后送测序。将重组质粒经限制性内切酶酶切制备成线性模板 ,通过体外转录的方法合成地高辛标记的mGluR1cRNA正义及反义探针。取成年Wistar大鼠小脑组织进行原位杂交实验 ,以检测探针的可靠性。结果 测序证实用RT PCR的方法获得了mGluR1基因特异片段 ,成功地构建了pGEM TmGluR1重组质粒。根据斑点杂交实验结果计算出正义、反义探针浓度分别为 1 0ng/ μl及 30ng/ μl。原位杂交实验的结果显示 ,用mGluR1反义探针进行杂交的阳性信号主要分布在大鼠小脑蒲肯野氏细胞胞浆 ,用正义探针杂交无阳性信号。结论 本实验克隆了mGluR1基因特异片段 ,并制备了cRNA探针 ,用大鼠小脑进行的原位杂交实验显示 ,此探针灵敏度高 ,特异性好
Objective To clone specific fragment of rat metabotropic glutamate receptor subtype 1 (mGluR1) and prepare cRNA probe. Methods The total RNA was extracted from the cerebellum of Wistar rats and the expected 599bp band was obtained by RT PCR. The fragment was cloned into pGEM Teasy vector and verified by restriction enzyme digestion. The recombinant plasmids were digested with restriction endonucleases to prepare a linear template. The digoxigenin-labeled mGluR1cRNA sense and antisense probes were synthesized by in vitro transcription. The adult Wistar rat cerebellum tissue was subjected to in situ hybridization to test the reliability of the probe. Results Sequencing confirmed that specific fragment of mGluR1 gene was obtained by RT PCR, and pGEM TmGluR1 recombinant plasmid was successfully constructed. According to the results of dot blot hybridization, the concentrations of sense and antisense probes were calculated as 10 ng / μl and 30 ng / μl, respectively. The results of in situ hybridization showed that the positive signals of hybridization with mGluR1 antisense probe mainly distributed in the cytoplasm of the Purkinje cell in the cerebellum and no positive signal was detected by the hybridization with the sense probe. Conclusion This study cloned mGluR1 gene specific fragment and prepared cRNA probe, in situ hybridization experiments using the rat cerebellum showed that this probe is highly sensitive and specific