siRNA-Mediated Gene Silencing in Neural Stem Cells

Protokół

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 incubation.
   3. Add an appropriate volume of siRNA-lipid complex to cells according to the manufacturer's recommendations.
   4. Incubate cells for 24 h in a standard cell culture incubator (37 °C, 5% CO₂). Perform downstream steps as appropriate (extract RNA, extract proteins, staining, etc.)
      NOTE: siRNA knockdown times and concentrations can vary depending on the individual experiment.

2. O9-1 cell differentiation

1. Differentiation of O9-1 cells into osteoblasts
   1. To prepare osteogenic differentiation media, dilute the following in alpha-MEM (final concentrations are indicated): 0.1 μM dexamethasone, 100 ng/mL bone morphogenetic protein 2 (BMP2), 50 µg/mL ascorbic acid, 10 mM b-glycerophosphate, 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin.
   2. Detect the osteoblast marker, osteocalcin, in differentiated osteoblasts by immunostaining, as shown in Figure 1.
      ​NOTE: Osteogenic differentiation can also be evaluated by using other markers of osteoblasts or with Alizarin red staining or alkaline phosphatase staining.
2. Differentiation of O9-1 cells into smooth muscle cells
   1. To prepare smooth muscle cell differentiation media, dilute the following in DMEM (final concentrations are indicated): 100 U/mL penicillin, 100 µg/mL streptomycin, and 10% FBS.
   2. Asses smooth muscle cell differentiation with immunofluorescence staining by using antibodies against markers of smooth muscle cells, such as smooth muscle actin (SMA), shown as an example in Figure 2.

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Wyniki

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), and nuclei were stained with DAPI (blue). Arrows indicate Ocn-positive cells. Osteocalcin (Ocn, an osteoblast marker); DAPI (′,6-diamidino-2-phenylindole).

Figure 2: Immunofluorescence staining of smooth muscle actin (SMA) indicating that, under smooth muscle cell differentiation conditions, most wild-type O9-1 cells gave rise to smooth muscle cells (A), whereas Yap-null O9-1 cells failed to differentiate into smooth muscle cells (B). Cells were stained with SMA antibody (red), and nuclei were stained with DAPI (blue). Arrows indicate SMA-positive cells.

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Materiały

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