Publications (2)0 Total impact
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ABSTRACT: 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.Lin chuang er bi yan hou ke za zhi = Journal of clinical otorhinolaryngology 08/2011; 25(16):751-5.
Article: [Construction of a recombinant eukaryotic vector of Atoh1 and its expression in 293-T cells].[show abstract] [hide abstract]
ABSTRACT: 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.Nan fang yi ke da xue xue bao = Journal of Southern Medical University 07/2011; 31(7):1131-7.