Effect Of Rac1 On Migration Of Neural Stem Cells

Neural stem cells (NSCs) can be defined as CNS progenitor cells that have the capacity for self-renewal and multi-potent potential to become neurons or glial cells. Migration of NSCs is critical for the development and repair of the nervous system. The tremendous migratory capability of NSCs in conjunction with their innate chemotropism for intracranial pathologies has been exploited for cell replacement therapies to treat diverse neurological diseases. Furthermore murine and human neural stem cells possess an inherent tumor tropism, represent a new and potentially powerful approach in the treatment of invasive tumours. Used as a delivery vehicle to target and disseminate therapeutic gene products throughout tumour sites, NSCs may overcome major obstacles facing current gene therapy strategies by infiltrating the tumour mass selectively and aggressively. A thorough understanding of the molecular events that regulate NSC migration to glioma is necessary to optimize the use of NSCs as therapeutic delivery vehicles.Members of the Rho family of small GTPases are key regulators of cell movement through their actions on actin assembly, actomyosin contractility, and microtubules. A vast majority of the studies involving Rho GTPases have been focused to the famous triad, Cdc42, Rac1 and RhoA, they cycle between two conformational states: one bound to GTP active state, the other bound to GDP inactive state. In neurons, these proteins have been proved crucial for migration. Therefore our goal is to figure out how these small GTPases effect the migration of NSCs.In our experiment, rat rac1 cDNA was cloned by RT-PCR method. TA clone was performed to insert the product into the pMD19-T vector. The recombinant plasmid was purified and used as a template. Three Rac1 mutants, constitutively active mutant Rac1-Q61L, Rac1-G12V and dominant negative mutant Rac1-T17N, were constructed by site-directed Mutagenesis method. Then Rac1 cDNA, Rac1-G12V and Rac1-T17N segments were cloned into a eukaryote plasmid pIRES2-EGFP by direct cloning after two restrictive endonucleases digestion. The recombinant vectors were verified by DNA sequence analysis.Sequences with 100% homology to rat Rac1 were generated using BLOCK-iT? RNAi Designer (Invitrogen). The target sequences showed no significant homology to other mouse proteins as determined by BLAST analysis. The sequence was used to generate oligonucleotide pairs, and inserted into the pcDNA6.2-GW/EmGFP-miR vector.At last four eukaryotic expression vectors(pIRES2-EGFP-Rac1, pcDNA6.2-GW/ EmGFP-miR2,pIRES2-EGFP-Rac1G12V, pIRES2-EGFP-Rac1T17N) we have constructed above were introduced into neural stem cell -like cell line C17.2 by transient transfections using Lipofectamine 2000 according to manufacturer's instructions with a transfection efficiency of 60–80%. To investigate the migration of cells, 24~48 h after transfection, phase-contrast images of C17.2 migrating randomly had been recorded by Leica DMI 6000 B for a period of 6 h. Cells which not contact with others from three independent experiments were chosen to assay FMI, the ratio of the effective distance over its total path length, and migration speed derived from the change in center position between subsequent images using Image J. The statistics result show that either down-regulation or up-regulation of Rac1 has no significantly change on the cell's migration speed and FMI. While the migration speed were significantly increased or decreased respectively in Rac1G12V and Rac1T17N transfected cells, but their FMI still have no significantly change.These result suggested that only up-regulation or down-regulation the expression of Rac1 would not significantly change the cell migration speed, for Rac1-GTP is the active form which can regulate cell migration. So the cell migration speed of Rac1G12V and Rac1T17N transfected cells had significantly increased or decreased respectively. The FMI indicates the direction migration level of the study cells, therefore either change in the expression of Rac1 or Rac1-GTP cannot significantly affect the cell's FMI. This study will contributes to better understanding of roles Rac1 played in the migration of NSCs, and would be helpful in the study of NSCs used as a gene deliver vector therapy for glioma.