Construction of CRMP4 lentiviral vector and its influence on neurite development
Chinese Journal of Clinical Anatomy ›› 2015, Vol. 33 ›› Issue (3) : 305-310.
Construction of CRMP4 lentiviral vector and its influence on neurite development
Objective This study aimed to investigate the influence of CRMP4 on the neurite growth in the hippocampal neuron. Methods To obtain the CRMP4 gene fragments by PCR amplification technology, then connect the gene to the viral vector. After PCR, enzyme restriction digestion and sequencing verification, the plasmids was packaged by transferring 293T cells to prepare Lentivirus Vector. The CRMP4 lentivirus vector was used to transfect hippocampal neurons cultured in vitro and neurite length of neurons was analyzed. Results The sequence of CRMP4 amplified by PCR was right and the gene fragment was inserted into lentivirus vector. The concentration of the virus was at the titer of 1.0×107~1.0×108 TU/ml. In the control cells, GFP was expressed in the neurites and cell bodies. Hippocampal neurons have two types of highly polarized cell processes, axons and dendrites with the development. When infected by CRMP4 Lentivirus Vector, the neurite outgrowth was obvious, and a lot of dendrites arose from cell bodies. The results of neurite lengths showed that over-expression CRMP4, the neutrite outgrowth was increased significantly and a lot of dendrites werefound. In cells without CRMP4 expression, the neutrites were few, and the length of neutritesdiminishedsignificantly when compared with CRMP4 over-expressing cells (P<0.05). Conclusion The lentivirus vector of CRMP4 was successfully constructed and overexpression of CRMP4 is beneficial to neurite development including neurite outgrowth and formation of branches.
CRMP4 / Lentivirus / Hippocampus / Neurons / Neurite
[1] Barzilai A, Zilkha-Falb R, Daily D, et al. The molecular mechanism of dopamine-induced apoptosis: identification and characterization of genes that mediate dopamine toxicity [J]. Neural Transm Suppl, 2000, (60):59-76.
[2] Hall C, Brown M, Jacobs T, et al. Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase [J]. Biol Chem, 2001, 276(46):43482-43486.
[3] Brot S, Rogemond V, Perrot V, et al. CRMP5 interacts with tubulin to inhibit neurite outgrowth, thereby modulating the function of CRMP2 [J]. Neurosci, 2010, 30(32): 10639-10654.
[4] Fukata Y, Itoh TJ, Kimura T, et al. CRMP-2 binds to tubulin heterodimers to promote microtubule assembly [J]. Nat Cell Biol, 2002, 4(8): 583-591.
[5] Katzourakis A, Tristem M, Pybus OG, et al. Discovery and analysis of the first endogenous lentivirus [J]. Proc Natl Acad Sci U S A, 2007, 104(15): 6261-6265.
[6] Cockrell AS, Kafri T. Gene delivery by lentivirus vectors [J]. Mol Biotechnol, 2007, 36(3): 184-204.
[7] Albright TD, Jessell TM, Kandel ER, et al. Neural science: a century of progress and the mysteries that remain [J]. Cell, 2000, 100 Suppl: S1-55.
[8] Lowery LA, Van Vactor D. The trip of the tip: understanding the growth cone machinery [J]. Nat Rev Mol Cell Biol, 2009, 10(5): 332-343.
[9] Geraldo S, Gordon-Weeks PR. Cytoskeletal dynamics in growth-cone steering [J]. Cell Sci, 2009, 122(Pt 20): 3595-3604.
[10]Yoshimura T, Kawano Y, Arimura N, et al. GSK-3beta regulates phosphorylation of CRMP-2 and neuronal polarity [J]. Cell, 2005, 120(1): 137-149.
[11] Rosslenbroich V, Dai L, Baader SL, et al. Collapsin response mediator protein-4 regulates F-actin bundling [J]. Exp Cell Res, 2005, 310(2): 434-444.
[12]Liu BP, Strittmatter SM. Semaphorin-mediated axonal guidance via Rho-related G proteins[J]. Curr Opin Cell Biol, 2001, 13(5): 619-626.
[13] Arimura N, Kaibuch K. Neuronal polarity: from extracellular signals to intracellular mechanisms[J]. Nat Rev Neurosci, 2007, 8(3): 194-205.
[14] Kowara R, Menard M, Brown L, et al. Co-localization and interaction of DPYSL3 and GAP43 in primary cortical neurons[J]. Biochem Biophys Res Commun, 2007, 363(1): 190-193.
/
| 〈 |
|
〉 |
