|
|
|
| Construction and Identification of a Lentiviral Vector Expressing Semaphorin 4D |
| SUN Qiang-ming, PAN Yue, ZHAO Yu-jiao, CHEN Jun-ying, SHI Hai-jing, MA Shao-hui |
| Institute of Medical Biology, Chinese Academy of Medical Sciences and Peking Union Medical College, Yunnan Key Laboratory of Vaccine Research & Development on Severe Infectious Diseases, Kunming 650118, China |
|
|
|
Abstract Objective: To construct a lentiviral vector expressing Semaphorin 4D to investigate the role of Semaphorin 4D in angiogenesis. Methods: Semaphorin 4D sequence was amplified from the Jurkat cell cDNA, inserted into pWPI GW vector and identified by DNA sequencing. The packing cell line (human embryonic kidney 293T cell) was cotransfected with pWPI GW/Sema4D, pVSVG and pSPAX. The recombinant lentivirus was packaged and amplified, followed by infection of 293T and HUVEC cells. The expression level of Semaphorin 4D gene was analyzed by Western blot and immunofluresence staining. A Boyden chamber migration assay was performed to establish the biological significance of Semaphorin 4D. Results: The recombinant lentiviral vector expressing Semaphorin 4D was successfully constructed. Western blot and immunofluresence staining results demonstrated that Semaphorin 4D was successfully expressed in 293T and HUVEC cells infected by recombinant lentivirus expressing Semaphorin 4D. Boyden chamber migration assay results showed that enhanced endothelial cell migration is induced by Sema4D-expressing Hela cells.
|
|
Received: 21 February 2011
Published: 25 July 2011
|
|
|
|
|
|
[1] Swiercz J M, Kuner R, Behrens J, et al. Plexin-B1 Directly Interacts with PDZ-RhoGEF/LARG to Regulate RhoA and Growth Cone Morphology. Neuron, 2002, 35(1):51-63.
[2] Conrotto P, Valdembri D, Corso S,et al. Sema4D induces angiogenesis through Met recruitment by Plexin B1. Blood, 2005, 105(11):4321-4329.
[3] Basile J R, Barac A, Zhu T, et al. Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B. Cancer Res, 2004, 64(15):5212-5224.
[4] Sierra J R, Corso S, Caione L, et al. Tumor angiogenesis and progression are enhanced by Sema4D produced by tumor-associated macrophages. J Exp Med, 2008, 205(7):1673-1685.
[5] Basile J R, Castilho R M, Williams V P, et al. Semaphorin 4D provides a link between axon guidance processes and tumor-induced angiogenesis. Proc Natl Acad Sci USA, 2006, 103(24):9017-9022.
[6] Sun Q M, Nawabi-Ghasimi F, Basile J R. Semaphorins in vascular development and head and neck squamous cell carcinoma-induced angiogenesis. Oral Oncol, 2008, 44(6):523-531.
[7] Matrai J, Chuah M K, VandenDriessche T. Recent advances in lentiviral vector development and applications. Mol Ther, 2010, 18(3):477-490.
[8] Tiscornia G, Singer O, Verma I M. Production and purification of lentiviral vectors. Nat Protoc, 2006, 1(1):241-245.
[9] Kolodkin A L, Matthes D J, Goodman C S. The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules. Cell, 1993, 75(7):1389-1399.
[10] Dittgen T, Nimmerjahn A, Komai S, et al.Lentivirus-based genetic manipulations of cortical neurons and their optical and electrophysiological monitoring in vivo. Proc Natl Acad Sci USA, 2004, 101(52):18206-18211.
[11] Cockrell A S, Kafri T.Gene delivery by lentivirus vectors. Mol Biotechnol, 2007, 36(3):184-204.
[12] Cartier N, Hacein-Bey-Abina S, Bartholomae C C, et al.Hematopoietic stem cell gene therapy with a lentiviral vector in X-linked adrenoleukodystrophy. Science, 2009, 326(5954):818-823.
|
|
Viewed |
|
|
|
Full text
|
|
|
|
|
Abstract
|
|
|
|
|
Cited |
|
|
|
|
| |
Shared |
|
|
|
|
| |
Discussed |
|
|
|
|
