Zheng Guoxi, Zhu Zhu, Zhu Kang, Hou Jin, Wei Junrong, Xu Min
Department of Otorhinolaryngology, the Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China.
Lin Chuang Er Bi Yan Hou Tou Jing Wai Ke Za Zhi. 2011 Aug;25(16):751-5.
To clone Atoh1 gene coding sequence of SD rat and construct the Eukaryotic expression plasmid pAtoh1-IRES2-EGFP,and to study its expression in 293T cells.
Total RNA was extracted from colon of SD rat. Atoh1 cDNA was obtained by RT-PCR amplification and subcloned into PMD-19T vector. The purified digested fragment was connected into Eukaryotic expression vector pIRES2-EGFP to construct the recombinant plasmid. The recombinant expression plasmid was identified by enzyme digestion and sequence analysis and then transfected into 293T cells with Lipofectamine. The expression of green fluorescent protein was detected through fluorescence microscope.
Compared cloned DNA sequence of Atoh1 gene CDS area with the reference sequences published in GeneBank, there were two base nonsense mutation in the sequence, deduced amino acid of cloning sequences as the same as reference sequences. Two bases should be single nucleotide polymorphism. Results of enzyme digestion and sequencing confirmed the successful construction of the recombinant plasmid. The expression of the green fluorescent protein was observed in the transfected 293T cells 24 h after transfection by fluorescence microscope.
pIRES2-EGFP-Atoh1 can be constructed and expressed successfully in the 293T cells, which will guide further research on gene therapy for sensorineural hearing loss.
克隆SD大鼠Atoh1基因编码序列，构建真核表达质粒pAtoh1-IRES2-EGFP，并研究其在293T细胞中的表达。
提取SD大鼠结肠组织总RNA。通过RT-PCR扩增获得Atoh1 cDNA，并亚克隆至PMD-19T载体。将纯化的酶切片段连接至真核表达载体pIRES2-EGFP，构建重组质粒。通过酶切和序列分析鉴定重组表达质粒，然后用Lipofectamine转染至293T细胞。通过荧光显微镜检测绿色荧光蛋白的表达。
将克隆的Atoh1基因CDS区DNA序列与GeneBank中公布的参考序列比较，序列中有两个碱基无义突变，克隆序列推导的氨基酸与参考序列相同。这两个碱基应为单核苷酸多态性。酶切和测序结果证实重组质粒构建成功。转染后24 h，通过荧光显微镜在转染的293T细胞中观察到绿色荧光蛋白的表达。
pIRES2-EGFP-Atoh1可成功构建并在293T细胞中表达，这将为感音神经性听力损失的基因治疗进一步研究提供指导。
