Zheng Guo-xi, Zhu Kang, Zhu Zhu, Hou Jin, Wei Jun-rong, Xu Min
Department of Otorhinolaryngology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710004, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2011 Jun;31(7):1131-7.
To clone the coding sequence of Atoh1 cDNA from SD rat and construct a eukaryotic expression vector for its expression in eukaryotic cells.
The total RNA was extracted from colonic mucosa of SD rats and the double-stranded cDNA of Atoh1 was amplified using RT-PCR. The cDNA coding sequence was then cloned into PMD-19T vector followed by sequence analysis. The digested fragment, after purification, was linked into the eukaryotic expression vector pIRES2-EGFP containing the EGFP gene and the internal ribosomes site (IRES). After identification by enzyme digestion and sequence analysis, the recombinant plasmid was transfected into 293T cells via Lipofectamine, and the expression of the target protein was detected under fluorescence microscope, using PT-PCR and Western blotting.
DNA sequence analysis showed that the amplified rat Atoh1 gene, with a length of 1056 bp of the coding sequence which encoded 351 amino acids, had two base mutations compared to the reference sequences in GenBank, possibly as a result of single nucleotide polymorphisms that induced nonsense mutation without affecting the amino acid sequences or the protein expression. The results of enzyme digestion and sequence analysis demonstrated that the Atoh1 gene was correctly inserted in the eukaryotic expression vector plRES2-EGFP. Fluorescence microscopy and Western blotting both confirmed successful expression of Atohl at the mRNA and protein levels in 293T cells 48 h after transfection with the recombinant plasmid.
The recombinant plasmid harboring the coding sequence of SD rat Atoh1 gene has been constructed successfully and can be expressed in the 293T cells, which provides a basis for functional study of Atoh1 gene and future researches in gene therapy for sensorineural hearing loss.
从SD大鼠中克隆Atoh1 cDNA的编码序列，并构建真核表达载体以在真核细胞中表达。
从SD大鼠结肠黏膜中提取总RNA，采用RT-PCR扩增Atoh1的双链cDNA。然后将cDNA编码序列克隆到PMD-19T载体中，随后进行序列分析。纯化后的酶切片段与含有EGFP基因和内部核糖体位点（IRES）的真核表达载体pIRES2-EGFP连接。经酶切鉴定和序列分析后，通过Lipofectamine将重组质粒转染至293T细胞中，并利用荧光显微镜、PT-PCR和Western印迹检测目的蛋白的表达。
DNA序列分析显示，扩增得到的大鼠Atoh1基因编码序列长度为1056 bp，编码351个氨基酸，与GenBank中的参考序列相比有两个碱基突变，可能是单核苷酸多态性导致的无义突变，但不影响氨基酸序列或蛋白质表达。酶切鉴定和序列分析结果表明，Atoh1基因已正确插入真核表达载体pIRES2-EGFP中。荧光显微镜和Western印迹均证实，重组质粒转染293T细胞48 h后，Atoh1在mRNA和蛋白水平均成功表达。
成功构建了携带SD大鼠Atoh1基因编码序列的重组质粒，且该质粒可在293T细胞中表达，为Atoh1基因的功能研究及未来感音神经性听力损失的基因治疗研究奠定了基础。
