Alteration of Differentiation Potentials by Modulating GATA Transcription Factors in Murine Embryonic Stem Cells
Figure 5
Expression array analysis and verification by quantitative RT-PCR of retinoic acid-induced endoderm differentiation of GATA-deficient ES cells. Wild type embryonic stem cells and those homozygous deficient in GATA4, GATA5, or GATA6 were cultured as monolayers and treated with the DMSO carrier as a control or retinoic acid (1 M) for 4 days. (a) Representative morphology of the cells with or without retinoic acid. (b) The cells were analyzed by cDNA expression microarray comparing with or without retinoic acid. The results were analyzed by hierarchical clustering. Individual colored rows represent change in expression following retinoic acid treatment of a single gene/sequence tag. Red rows indicate an increase in expression and green rows indicate a decrease in expression, as shown by the color scale bar. (c)–(h) Verification of expression of a panel of selected genes by quantitative RT-PCR Relative gene expression changes with or without retinoic acid in monolayer cells (c) and spheroids (d) are presented as “Heat-Maps”. The figure displays grey scale shades representing the Ct values. Ct (cycle threshold) is the number of PCR cycles at which the fluorescence reaches a significant level above the baseline, given that the higher the starting copy number of the nucleic acid target, the sooner fluorescence increases. The relative levels of a particular transcript between samples can be calculated using the equation: relative quantity 2 − Ct. The amplification of TBP shows similar amounts of template in all samples. (e)–(h) values that represented relative mRNA levels of the monolayer ES cells are shown and compared. The mRNA values of undifferentiated ES cells are defined as “1” for comparison. The detection of a real-time RT-PCR signal in a specific GATA transcript in the ES cells that were homozygous knockout of that GATA gene is likely due to the presence of transcripts from the mutant/inactive GATA locus.
