Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors

China Biotechnology

2011, Vol. 31

Issue (5): 1-7 DOI:

Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors

GUO Shu-jun¹, WAN Yan¹, LI Li-ling^1,2, QING Li¹, CHEN Xiao-jia¹

1. National Engineering Research Center of Genetic Medicine, Guangdong Provincial Key Laboratory of Bioengineering Medicine, Bioengineering Institute of Jinan University,Guangzhou 510632, China;
2. Shenzhen Nanshan Chronic Diseases Prevention and Cure Center, Shenzhen 518000,China

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Abstract

Objective: To establish the recombinant L6 myoblasts with exogenous FGFR2IIIc by lentivirus and to distinguish their characters. Methods: FGFR2IIIc gene was obtained by RT-PCR from human placental and was cloned into pENTA-11. Recombinant expression vector pLenti6/V5-DEST-FGFR2-IIIc was obtained by using pENTA-FGFR2IIIc and pLenti6/V5-DEST plasmids with LR recombinase. Then such vector and Viral Packaging Mix vectors were co-transfected into 293FT cells to gain recombinant lentiviral particles. After infected by Lentivirus,stable L6 cell monoclones were selected by blasticidin for 2~4 weeks. The recombinant cell lines with exogenous FGFR2IIIc were identified by RT-PCR,indirect immunofluorescence and Western blot. Co-stimulated by both bFGF and HSPG, cell cycles of recombinant L6 cells were evaluated by flow cytometry. Their myogenic differentiation was observed with or without inhibitors of ERK and p38 signal pathway. Results: The vector pLenti6/V5-DEST-FGFR2-IIIc was reconstructed to gain lentiviral particles to establish recombinant L6 myoblasts with exogenous FGFR2IIIc.The recombinant L6-FGFR2IIIc group show an increase in the percentage of cells in the G1/G0 phase compared to the L6-vector group. Phosphorylated Erk1/2 inhibition with PD98059 leads to some cells myogenesis;And phosphorylated p38 inhibition with SB203580 leads to cells apoptosis. Conclusions: FGFR2IIIc can inhibit L6 myoblasts proliferation and result in the tendency of myogenic differentiation.

Key words： FGFR2IIIc Lentivirus expression system L6 myoblasts

Received: 26 November 2010 Published: 27 May 2011

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GUO Shu-jun, WAN Yan, LI Li-ling, QING Li, CHEN Xiao-jia. Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors. China Biotechnology, 2011, 31(5): 1-7.

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https://manu60.magtech.com.cn/biotech/ OR https://manu60.magtech.com.cn/biotech/Y2011/V31/I5/1

[1] Luo Y, Lu W,Mohamedali K A,et al.The glycine box:a determinant of specificity for fibroblast growth factor.Bioehemistry,1998,37(47):16506-16515.

[2] Dionne C A,Crumley G,Bellot F,et al.Cloning and expression of two distinct high-affinity receptors cross-reacting with acidic and basic FGFs. EMBO J, 1990,9(9):2685-2692.

[3] Miki T,Fleming T P,Bottaro D P,et al.Expression cDNA cloning of the KGF receptor by creation of a transforming autocrine loop.Science, 1991,251(4989):72-75.

[4] Katoh M,Hattori Y,Tanaka M,et al.K-sam gene encodes secreted as well as transmembrane receptor tyrosine kinase.Proc Natl Acad Sci USA, 1992,89(7):2960-2964.

[5] Ornitz D M,Xu J,Colvin J S,et al.Receptor specificity of the FGF family.J Biol Chem, 1996, 271(25): 15292-15297.

[6] Zhang X,Ibrahimi O A,Olsen S K,et al.Receptor specificity of the FGF family.J Biol Chem, 2006,281(23):15694-15700.

[7] Delezoide A L,Benoist-Lasselin C,Lefeai-Mallet L,et al.Spatio-temporal expression of FGFR 1,2and 3 genes during human embryo-fetal ossification.Mech Dev,1998,77(1):19-30.

[8] Cohen M M, Kreiborg S.The central nervous system in the Apert syndrome .Am J Med Genet, 1990, 35(1):36-45.

[9] Hatano N,Mori Y,Oh-hora M,et al.Essential role for ERK2 mitogen-activated protein kinase in placental development.Genes Cells,2003,8(11):847-856.

[10] Saba-El-Leil M K,Vella F D,Vernay B,et al.An essential function of the mitogen-activated protein kinase Erk2 in mouse trophoblast development.EMBO Rep,2003,4(10):964-968.

[11] Yao Y, Li W,Wu J,et al.Extracellular signal-regulated kinase 2 is necessary for mesoderm Differentiation.Proc Natl Acad Sci USA,2003,100(22):12759-12764.

[12] Jones N C,Fedorov Y V,Rosenthal R S,et al.ERK1/2 is required for myoblast proliferation but is dispensable for muscle gene expression and cell fusion.J Cell Physiol,2001,186(1):104-115.

[13] Nebreda A R,Porras A. p38 MAP kinases:beyond the stress response.Trends Biochem Sci,2000,25(6):257-260.

[14] Zarubin T,Han J.Activation and signaling of the p38 MAP kinase pathway.Cell Res,2005,15(1):11-18.

[15] Natalicchio A,Laviola L,De Tullio C D,et al.Role of the p66shc isoform in insulin-like growth factor I receptor signaling through MEK/Erk and regulation of Actin Cytoskeleton in rat Myoblasts. J Biol Chem, 2004, 279(42):43900-43909.

[16] Penn B H,Bergstrom D A,Dilworth F J,et al.A MyoD-generated feed-forward circuit temporally patterns gene expression during skeletal muscle differentiation.Genes Dev,2004,18(19):2348-2353.

[17] Li J,Johnson S E.ERK2 is required for efficient terminal differentiation of skeletal myoblasts.BBRC,2006,345(4):1425-1433.

[18] Coolican S A,Samuel D S,Ewton D Z,et al.The Mitogenic and Myogenic Actions of Insulin-like Growth Factors Utilize Distinct Signaling Pathways. J Biol Chem, 1997,272(10):6653-6662.

[19] Wu Z,Woodring P J,Bhakta K S,et al. p38 and extracellular signal-regulated kinases regulate the myogenic program at multiple steps.Mol Cell Biol,2000,20(11):3951-3964.

[20] Zetser A,Gredinger E,Bengal E. p38 mitogen-activated protein kinase pathway promotes skeletal muscle differentiation.Participation of the Mef2 transcription factor. J Biol Chem, 1999,274(8):5193-5200.

[21] Katoh Y, Katoh M. FGFR2-related pathogenesis and FGFR2-targeted therapeutics. International Journal of Molecular Medicine, 2009, 23(3):307-311.

[22] Arman E, Haffner-Krausz R, Gorivodsky M, et al. Fgfr2 is required for limb outgrowth and lung-branching morphogenesis.Proc Natl Acad Sci USA,1999,96(21):11895-11899.

[23] Miraoui H, Severe N, Vaudin P, et al. Molecular silencing of Twist1 enhances osteogenic differentiation of murine mesenchymal stem cells:implication of FGFR2 signaling. Journal of Cellular Biochemistry, 2010, 110(5):1147-1154.

[24] Walsh K, Perlman H. Cell cycle exit upon myogenic differentiation. Current Opinion in Genetics & Development, 1997, 7(5):597-602.

[25] Esko J D,Selleck S B. Order out of chaos: assembly of ligand binding sites in heparan sulfate. Ann Rev Biochem, 2002, 71:435-471.

[26] Kan M, Wang F, Xu J, et al. An essential heparin-binding domain in the fibroblast growth factor receptor kinase. Science, 1993, 259(5103):1918-1921.

[27] Gospodarowicz D,Cheng J. Heparin protects basic and acidic FGF from inactivation. J Cell Physiol, 1986, 128(3):475-484.

[28] Vanden-Driessche T,Vanslembrouck V,Goovaerts I,et al.Long-term expression of human coagulation factor Ⅷ and correction of hemophilia A after in vivo retroviral gene transfer in factor Ⅷ-deficient mice.Proc Natl Acad Sci U S A, 1999, 96(18):10379-10384.

[29] Connelly S,Smith T A,Dhir G,et al.In vivo delivery and expression of physiological levels of functional human factor Ⅷ in mice.Hum Gene Ther, 1995, 6(2):185-193.

[30] Naldini L,Blomer U,Gallay P,et al.In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector.Science,1996,272(5259):263-267.

[31] Kafri T,Blomer U,Peterson D A,et al.Sustained expression of genes delivered directly into liver and muscle by lentiviral vectors.Nat Genet,1997,17(3):314-317.

[1]	. Expression of Human FGFR2IIIc in L6 with Lentivirus Vectors[J]. China Biotechnology, 2011, 31(05): 0-0.

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