|Public on May 22, 2012
|cell type: ZiNPC derived from BJ1 fibroblasts
cell line: BJ1
treatment: Zic3, KLF4, OCT4 and SOX2
|The coding region of the mouse Zic3 gene was amplified by RT-PCR and cloned in pMIG-IRES-GFP plasmid (Addgene). The pMXs plasmids encoding for OCT4, SOX2 and KLF4 were purchased from Addgene. pMXs and pMIG-based retroviral vectors were transfected individually along with the viral packaging genes gag-pol (Addgene) and VSVG (Addgene) into 293T cells (ATCC) using Fugene HD reagent (Roche) according to the manufacturer's directions. Human BJ1 cells (Lonza) were seeded at 1.5 x 105 cells/well in 6-well plates (Sigma) without feeders. Cells were transduced with Zic3, KLF4, OCT4 and SOX2 retroviral vector containing supernatants mixed equally. To achieve maximum transduction efficiency, cells were transduced twice with the viral supernatant. Four days after transduction, the transduced cells were reseeded at 1.5 x 105 cells per 100-mm dish containing MEF and cultured with hESC medium. ZiNPC were picked in 15-20 days post transduction.
|RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Using the Ambion WT Expression Kit, per sample, an amount of 100 ng of total RNA spiked with bacterial poly-A RNA positive controls (Affymetrix) was converted to double stranded cDNA in a reverse transcription reaction. Next the sample was converted and amplified to antisense cRNA in an in vitro transcription reaction which was subsequently converted to single stranded sense cDNA. Finally, samples were fragmented and labeled with biotin in a terminal labeling reaction according to the Affymetrix WT Terminal Labeling Kit.
|A mixture of fragmented biotinylated cDNA and hybridisation controls (Affymetrix) was hybridised on Affymetrix GeneChip Human Gene 1.1 ST Array Plate followed by staining and washing in the GeneTitan® Instrument (Affymetrix) according to the manufacturer's procedures.
|To assess the raw probe signal intensities, chips were scanned using the GeneTitan® HT Array Plate Scanner (Affymetrix).
|BJ1 fibroblast transduced with OCT4, SOX2, KLF4 and Zic3
|RMA-normalized expression values as obtained with the xps package (version 1.10.2) of BioConductor
|Jun 07, 2011
|Last update date
|May 23, 2012
|Herestraat 49 Box 816
|Transduction of human fibroblasts with Zic3 combined with OCT4, SOX2 and KLF4 induces stable neural progenitor cell lines