title: [Construction of a recombinant eukaryotic vector of Atoh1 and its expression in 293-T cells].
abstract: OBJECTIVE
  To clone the coding sequence of Atoh1 cDNA from SD rat and construct a eukaryotic expression vector for its expression in eukaryotic cells.
  
  
  METHODS
  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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Otorhinolaryngology, Second Affiliated Hospital, Medical School of Xi'an Jiaotong University, Xi'an 710004, China. zhengguoxi@21cn.com
authors.forename: Guo-xi
authors.initials: GX
authors.lastname: Zheng
authors.forename: Kang
authors.initials: K
authors.lastname: Zhu
authors.forename: Zhu
authors.initials: Z
authors.lastname: Zhu
authors.forename: Jin
authors.initials: J
authors.lastname: Hou
authors.forename: Jun-rong
authors.initials: JR
authors.lastname: Wei
authors.forename: Min
authors.initials: M
authors.lastname: Xu
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000096:biosynthesis* / Q000235:genetics; D002460:Cell Line; D003001:Cloning, Molecular; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D051381:Rats; D017207:Rats, Sprague-Dawley; D011994:Recombinant Proteins / Q000096:biosynthesis* / Q000235:genetics; D014162:Transfection
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D012333:RNA, Messenger; D011994:Recombinant Proteins