Construction of eukaryotic expression vectors of two NF2 mutants and their expression in rat schwannoma cell line
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Supported by Foundation of Shanghai Science and Technology Committee(04qmh1414)and Natural Science Foundation of Shanghai(06ZR14064).

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    Abstract:

    Objective:To construct the eukaryotic expression vectors of two NF2 mutants (neurofibromatosisⅡ) and to study their expression in rat schwannoma cell line (RT4).Methods: Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Lys42 in NF2 into Pro in pcDNA3.1-NF2ΔLys42Pro, and convert the codons for Phe 47 into that of Leu.RT4 cells were transiently transfected with the 3 kinds of plasmids containing the mutations and wide-type NF2 via lipofectin separately,then the expression levels of NF2 mRNA and protein were determined by RT-PCR and Western blotting in 3 groups.The cell proliferation was determined by the MTT after transfection.Results: DNA sequence analysis demonstrated the two-step mutagenesis was successful and the two plasmids of pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu were obtained,both of the transfectants could produce merlin protein and mRNA.The two mutants had a significantly lower inhibitory rate for RT4 cells compared with wide-type NF2 (P<0.05).Conclusion: The recombinant plasmids pcDNA3.1-NF2ΔLys42Pro and pcDNA3.1-NF2ΔPhe47Leu have been successfully constructed and they can be efficiently expressed in RT4 cells.

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History
  • Received:March 05,2008
  • Revised:April 11,2008
  • Adopted:April 16,2008
  • Online: May 04,2008
  • Published:
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