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[The cloning and sequencing of SD rat Atoh1 gene CDS region]
Guoxi Zheng1, Zhu Zhu, Kang Zhu
1Department of Otorhinolaryngology, the Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an, 710004, China. zhengguoxi@21cn.com
Objective:
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.
Method:
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.
Result:
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.
Conclusion:
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.
