siRNA-Mediated Gene Silencing in Neural Stem Cells

Abstract

Source: Nguyen, B. H., et al. Culturing and Manipulation of O9-1 Neural Crest Cells. J. Vis. Exp (2018)

This video demonstrates the use of small interfering RNA (siRNA) to silence specific genes of interest in neural stem cells and differentiate them into desired cell types using appropriate differentiation medium. This procedure enables the study of the critical role of specific genes in the differentiation process of neural stem cells.

Protocol

1. Manipulation of O9-1 cells

1. Performing siRNA knockdown in O9-1 cells
   1. Thaw and coat a 24-well plate with a basement membrane matrix before starting the experiment. Recover and seed O9-1 cells, allowing them to grow to 60% to 80% confluency.
   2. Dilute liposomes in serum-free media according to the appropriate well volume. Dilute siRNA in serum-free media to the final. Add diluted siRNA to diluted liposomes in a ratio recommended by the manufacturer. Mix well by pipetting and then incubate.
      NOTE: Follow the manufacturer's guide on the volume used, time, and temperature of incubat....

Results

Figure 1: Immunofluorescence staining of osteoblast marker osteocalcin (Ocn) indicating that O9-1 cells gave rise to osteoblast cells under osteogenic differentiation conditions. Cells were stained with osteoblast marker Ocn antibody (green), a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
O9-1 mouse cranial neural crest cell line	Millipore Sigma	SCC049
DMEM, high glucose, no glutamine	Gibco	11960-044
DMEM, high glucose	Hyclone	SH30243.01
FBS (fetal bovine serum)	Millipore Sigma	ES-009-B
Penicillin - streptomycin	Gibco	15140-122
L-glutamine 200mM (100X)	Gibco	25030-081
Trypsin-EDTA 0.25% in HBSS	Genesee Scientific	25-510
DPBS (Dulbecco's phosphate buffered saline) without calcium or magnesium	Lonza	17-512F
MEM non-essential amino acids (MEM NEAA) 100X	Gibco	11140-050
Sodium pyruvate (100mM)	Gibco	11360-070
2-Mercaptoethanol	Sigma	M-7522
Recombinant human fibroblast growth factor-basic (rhFGF-basic)	R&D Systems	233-FB-025
Mitomycin C	Roche	10107409001
Matrigel matrix	Corning	356234
DMSO (dimethylsulfoxide)	Millipore Sigma	MX1458-6
Lipofectamine RNAiMAX	Thermo Fisher Scientific	13778-075
Opti-MEM I (1X)	Gibco	31985-070
Minimum essential medium, alpha 1X with Earle's salts, ribonucleosides, deoxyribonucleosides, & L-glutamine	Corning	10-022-CV
ON-TARGETplus Wwtr1 siRNA	Dharmacon	L-041057
ON-TARGETplus Non-targeting Pool	Dharmacon	D-001810
ON-TARGETplus Yap1 siRNA	Dharmacon	L-046247
FCS (fetal calf serum)
ITS (insulin-transferrin-selenium )
TGF-b3
Ascorbic acid
BMP2 (bone morphogenetic protein 2)
Dexamethasone
B-27 supplement