Construction and characterization of mutants in the neurofibromatosis type 2 tumor suppressor gene

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HE Hua(Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200433, China)
ZHAO Jian-xiang()
LU Yi-cheng(Department of Neurosurgery, Changzheng Hospital, Second Military Medical University, Shanghai 200433, China)
SUN Qing-fang()
BIAN Liu-guan()
SHEN Jian-kang()
Journal Title:
Volume 8, Issue 12, 2009
Key Word:
Site-directed mutagenesis; Eukaryotic expression vector; Neurofibromatosis type 2; Tumor suppressor gene; Rat schwannoma cell

Abstract: Objective To construct the eukaryotic expression vector of mutants in the neurofibromatosis type 2 (NF2) tumor suppressor gene and observe its expression in rat schwannoma cell line RT4. Methods Site-directed mutagenesis was performed to induce the mutation of the codons for the residue Ile 546 in pEGFP-N1-NF2 into Met to construct the muton of pEGFP-N1-NF2~(△Ⅱe546Met). After lipofectin-mediated transient transformation of RT4 with the plasmids containing the mutation and the one without mutation, respectively, the mRNA and protein expression levels of NF2 were determined using fluorescence imaging and Western blotting. The cell proliferation was determined by the MTT assay. Results DNA sequence analysis confirmed the success of site-directed mutagenesis and Western blotting showed that pEGFP-NF2 protein could be expressed in the RT4 cells. The RT4 cell inhibition rate in the pEGFP-N1-NF2~(△Ⅱe546Met) transfection group was statistically lower than that in the pEGFP-N1-NF2 transfected group (P<0.05).Conclusion The recombinant plasmids pEGFP-N1-NF2~(△Ⅱe546Met) has been successfully constructed with efficient expressions in RT4.

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