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Construction and Identification of cnksr2 Gene Interference Adenovirus(PDF)


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Construction and Identification of cnksr2 Gene Interference Adenovirus
Liang Jin1Shen Ning1Xu Kang2Li Chaojun1Xue Bin1
(1.Medical School of Nanjing University,Jiangsu Key Laboratory for Molecular Medicine,Nanjing 210093,China) (2.School of Life Sciences,Nanjing Normal University,Jiangsu Key Laboratory for Molecular and Medical Biotechnology,Nanjing 210023,China)
To construct a recombinant adenovirus vector expressing small RNA against mouse cnksr2 gene,the interferce primer were designed according to cnksr2 gene sequence,which was cloned into pshuttle-H1 vector with Bgl Ⅱ and Hind Ⅲ restriction site.E.coli BJ5183 sensitive bacterias were cotransfected with lined vector cutted by Pme I enzyme and adenovirus vector pAdEasy-1.The obtained recombinant adenovirus vector was cutted with Pac Ⅰ enzyme,then adenovirus was obtained in 293A cells transfected with lined recombinant adenovirus plasmids.The titer of virus was measured based on the appearing of CPE and was determined by TCID50 assay.The interference efficiency was caculated.The recombinant adenovirus vector targeting to cnksr2 gene was constructed as evidenced by DNA sequencing and identification of enzyme digestion,which titer of concentrated virus was 3.98×107 PFU/mL.The expression of endogenous cnksr2 mRNA in PC12 cells was specificially shutdown by the recombinant adenovirus vector with an efficiency above 50%.The interference of cnksr2 gene was successfully constructed.


[1] Therrien M,Wong A M,Rubin G M.CNK,a RAF-binding multidomain protein required for RAS signaling[J].Cell,1998,95(3):343-353.
[2]Nagase T,Ischikawa K,Suyama M,et al.Prediction of the coding sequences of unidentified human genes.Ⅻ.The complete sequences of 100 new cDNA clones from brain which code for large proteins in vitro[J].DNA Res,1998,5(6):355-364.
[3]Yao I,Hata Y,Ide N,et al.MAGUIN,a novel neuronal membrane-associated guanylate kinase-interacting protein[J].J Biol Chem,1999,274(17):11 889-11 896.
[4]Roy F,Iaberge G,Douziech M,et al.KSR is a scaffold required for activation of the ERK/MAPK module[J].Genes Dev,2002,16(4):427-438.
[5]Bumeister R,Rosse C,Anselmo A,et al.CNK2 couples NGF signal propagation to multiple regulatory cascades driving cell differentiation[J].Curr Biol,2004,14(5):439-445.
[6]Santos S D,Verveer P J,Bastiaens P I.Growth factor-induced MAPK network topology shapes Erk response determining PC-12 cell fate[J].Nat Cell Biol,2007,9(3):324-330.
[7]Ohtakara K,Nishizawa M,Izawa I,et al.Densin-180,a synaptic protein,links to PSD-95 through its direct interaction with MAGUIN-1[J].Genes Cells,2002,7(11):1 149-1 160.
[8]Eastwood S L,Burnet P W,Harrison P J.Altered synaptophysin expression as a marker of synaptic pathology in schizophrenia[J].Neuroscience,1995,66(2):309-319.
[9]Deakin J F,Simpson M D.A two-process theory of schizophrenia:evidence from studies in post-mortem brain[J].J Psychiatr Res,1997,31(2):277-295.
[10]Houge G,Rasmussen I H,Hovland R.Loss-of-function CNKSR2 mutation is a likely cause of non-syndromic X-linked intellectual disability[J].Mol Syndromol,2012,2(2):60-63.
[11]Vaudry D,Stork P J,Lazarovici P,et al.Signaling pathways for PC12 cell differentiation:making the right connections[J].Science,2002,296(5573):1 648-1 649.
[12]Pearson G,Robinson F,Beers Gibson T,et al.Mitogen-activated protein(MAP)kinase pathways:regulation and physiological functions[J].Endocr Rev,2001,22(2):153-183.
[13]Therrien M,Wong A M,Kwan E,et al.Functional analysis of CNK in RAS signaling[J].Proc Natl Acad Sci U S A,1999,96(23):13 259-13 263.
[14]Douziech M,Sahmi M,Laberge G,et al.A KSR/CNK complex mediated by HYP,a novel SAM domain-containing protein,regulates RAS-dependent RAF activation in Drosophila[J].Genes Dev,2006,20(7):807-819.
[15]Roignant J Y,Hamel S,Janody F,et al.The novel SAM domain protein Aveugle is required for Raf activation in the Drosophila EGF receptor signaling pathway[J].Genes Dev,2006,20(7):795-806.
[16]Friedman A,Perrimon N.A functional RNAi screen for regulators of receptor tyrosine kinase and ERK signalling[J].Nature,2006,444(7116):230-234.
[17]Anselmo A N,Burneister R,Thomas J M,et al.Critical contribution of linker proteins to Raf kinase activation[J].J Biol Chem,2002,277(8):5 940-5 943.
[18]Yao I,Ohtsuke T,Kawabe H,et al.Association of membrane-associated guanylate kinase-interacting protein-1 with Raf-1[J].Biochem Biophys Res Commun,2000,270(2):538-42.


Last Update: 2013-09-30