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

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Author:
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:
CHINESE JOURNAL OF NEUROMEDICINE
Issue:
Volume 8, Issue 12, 2009
DOI:
10.3760/cma.j.issn.1671-8925.2009.12.001
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.

  • [1]Eddleman CS,Liu JK.Optic nerve sheath meningioma:current diagnosis and treatment[J].Neurosurg Focus,2007,23(5):E4.
  • [2]Schefler AC,Dubovy SR,Berrocal AM.Optical coherence tomography characteristics of epiretinal membranes in neurofibromatosis 2[J].Ophthalmic Surg Lasers Imaging,2008,39 (1):73-77.
  • [3]Sestini R,Bacci C,Metvenzano A,et al.Evidence of a four-hit mechanism involving SMARCB1 and NF2 in schwannomatosis-associatcd schwannomas[J].Hum Mutat,2008,29(2):227-231.
  • [4]Jacoby LB,Jones D,Davis K,et al.Molecular analleis of the NF2 tumor-suppressor gene in schwannomatosis[J].Am J Hum Genet,1997,61(6):1293-1230.
  • [5]Fisher LM,Doherty JK,Lev MH,et al.Distribution of nonvestibular cranial nerve schwannomas in neurofibromatosis 2[J].Otol Neurotol,2007,28(8):1083-1090.
  • [6]Evans DG,Ramsden RT,Shenton A,et al.What are the implications in individuals with unilateral vestibular schwannoma and other neurogenic tumors?[J].J Neurosurg,2008,108(1):92-96.
  • [7]卞留贯,孙青芳,沈建康,等.神经鞘瘤的NF2基因2、4、6、13外显子突变分析[J].中国神经精神疾病杂志,2001,27:401-404.
  • [8]Bian LG,Tirakotai W,Sun QF,et al.Molecular genetics alterations and tumor behavior of sporadic vestibular schwaunoma from the People's Republic of China[J].J Neurooncol,2005,73(3):253-260.
  • [9]沈建康,卞留贯,孙青芳,等.神经鞘瘤的NF2基因突变分析[J].中华神经外科杂志,2002,18:96-99.
  • [10]贺子建,王晓强,卞留贯,等.NF2基因及其蛋白merlin的生物学研究进展[J].国际神经病学神经外科学杂志,2006,33:194-197.
  • [11]贺子建,卞留贯,贺华,等.野生型Ⅱ型神经纤维瘤病基因Ⅱ亚型真核表达载体的构建及其功能[J].中国现代神经疾病杂志,2007,7:305-309.
  • [12]Shimizu T,Seto A,Maita N,et al.Structural basis for neurofibromatosis type 2.Crystal structure of the merlin FERM domain[J].J Biol Chem,2002,277:10332-10336.
  • [13]Schulze KM,Hanemann CO,Muller HW,et al.Transduction of wild-type merlin into human schwannoma cells decreases schwannoma cell growth and induces apoptosis[J].Hum Mol Genet,2002,11:69-76.
  • [14]Nakai Y,Zheng Y,MacCollin M,et al.Temporal control of Rac in Schwann cell-axon interaction is disrupted in NF2-mutant schwannoma cells[J].J Neurosci,2006,26:3390-3395.
  • [15]Shaw RJ,Paez JG,Curto M,et al.The Nf2 tumor suppressor,merlin,functions in Rac-dependent signaling[J].Dev Cell,2001,1:63-72.
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