Laboratory of Molecular Carcinogenesis, National Institute of Environmental Health Sciences
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Zheng, X., Hu, G. Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal. J. Vis. Exp. (63), e3987, doi:10.3791/3987 (2012).
Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will greatly facilitate the use of ES cells for developmental biology studies, disease modeling, drug discovery, and regenerative medicine (reviewed in 1,2).
To expedite the identification and characterization of novel regulators of ES cell maintenance and self-renewal, we developed a fluorescence reporter-based assay to quantitatively measure the self-renewal status in mouse ES cells using the Oct4GiP cells 3. The Oct4GiP cells express the green fluorescent protein (GFP) under the control of the Oct4 gene promoter region 4,5. Oct4 is required for ES cell self-renewal, and is highly expressed in ES cells and quickly down-regulated during differentiation 6,7. As a result, GFP expression and fluorescence in the reporter cells correlates faithfully with the ES cell identity 5, and fluorescence-activated cell sorting (FACS) analysis can be used to closely monitor the self-renewal status of the cells at the single cell level 3,8.
Coupled with RNAi, the Oct4GiP reporter assay can be used to quickly identify and study regulators of ES cell maintenance and self-renewal 3,8. Compared to other methods for assaying self-renewal, it is more convenient, sensitive, quantitative, and of lower cost. It can be carried out in 96- or 384-well plates for large-scale studies such as high-throughput screens or genetic epistasis analysis. Finally, by using other lineage-specific reporter ES cell lines, the assay we describe here can also be modified to study fate specification during ES cell differentiation.
1. Oct4GiP Mouse ES Cell Maintenance
Oct4GiP cells were kindly provided by Dr. Austin Smith. They were derived from the 129/Ola mice carrying an Oct4-GFPiresPac transgene 4,5 . They are maintained in gelatin-coated tissue culture plates in the ESGRO complete plus clonal grade medium (Millipore), or in the M15 medium: DMEM (Invitrogen) supplemented with 15% FBS, 1000 U/ml ESGRO (Millipore), 1x Non-essential amino acids (Invitrogen), 1x EmbryoMax Nucleasides (Millipore), and 10 μM β-mercaptoethanol.
2. siRNA Transfection in Oct4GiP Cells
The Oct4GiP reporter assay is most conveniently carried out in gelatin-coated flat bottom 96- or 384-well plates (Corning or BD) in the M15 medium. The overall procedure is outlined in Figure 1.
Note: The final siRNA concentration in the transfections is 50 nM. Carry out 3-4 biological replicates for each siRNA transfection. Set up master mixtures of the siRNA-lipid complexes in the U-bottom plate and aliquot into the flat-bottom gelatin-coated plate.
Note: The optimal cell plating density is usually 3 x 105 cells/cm2, but it may require further optimization for siRNAs that dramatically affect cell growth or viability. Low plating density will lead to poor cell survival during transfection. High plating density will lead to high background due to high cell confluence induced differentiation. If necessary, test plating density between 2-4 x 105 cells/cm2.
3. FACS Analysis
4. Representative Results
Oct4, Nanog, and Sox2 are three genes that play critical roles in the maintenance of ES cell self-renewal 6,7,9-11. Figure 2 shows that the Oct4GiP reporter assay can readily detect the differentiation caused by silencing these factors in the Oct4GiP ES cells.
Figure 2A shows the Oct4GiP ES cells are GFP-positive when maintained as ES cells. Figure 2B shows the forward vs. side scatter plot and the histogram of the GFP channel of the Oct4GiP cells transfected with the control- or Oct4-siRNAs. It is necessary to gate for live cells in the forward vs. side scatter plot, as the dead cells and debris are GFP-negative and will increase background. On the other hand, doublet discrimination to exclude possible cell clumps is not always needed. In the control-siRNA transfected cells, the vast majority of the cells should be GFP-positive. If obvious GFP-negative populations are present in the control-siRNA transfected wells, the starting Oct4GiP cells or the transfection procedure may have been compromised and the result may not be interpretable.
Figure 2C shows the bar graph of % Differentiated cells (% Differentiated cells = % GFP-negative cells) from control-, Oct4-, Nanog-, and Sox2-siRNA transfected cells.
Figure 1. Outline of the Oct4GiP reporter assay.
Figure 2. Oct4GiP reporter assay can detect ES cell differentiation caused by Nanog, Oct4, or Sox2 silencing. A) E14Tg2a (wild-type, black line) and Oct4GiP (green line) cells were analyzed by FACS. Histogram of the GFP-channel shows that Oct4GiP cells are GFP-positive. B) Oct4GiP cells were transfected in 96-well plates with the Control- or Oct4-siRNA and FACS analyzed 4 days after transfection. Forward vs. side scatter plots and histograms of the GFP-channel of the transfected cells are shown. C) Oct4GiP cells were transfected with the Control-, Nanog-, Oct4-, or Sox2-siRNA. The % Differentiated Cells was determined from the % GFP-negative cells 4 days after transfection, and was plotted as mean +/- standard error of the mean (n = 4). Click here to view larger figure.
The Oct4GiP reporter assay we describe above can quantitatively measure the extent of self-renewal vs. differentiation. Compared to other available methods, such as the morphology-based 12 and proliferation/viability-based assays, it offers higher sensitivity and throughput, as well as a more direct measurement of the ES cell state. It is therefore well suited for large-scale screens and genetic epistasis analysis. Indeed, we and others have successfully used the Oct4GiP reporter assay for genome-wide RNAi screens 3,8. Like all assays, however, it also has limitations. It can only be used to study genes that directly or indirectly regulate Oct4 promoter activity, and it may falsely identify genes that affect GFP expression, stability, or function post-transcriptionally. Therefore, additional assays or secondary screens, such as alkaline phosphatase staining 13 (Millipore, SCR004) and immunofluorescence staining or quantitative RT-PCR of pluripotency markers 3,8,12,14 are required to confirm results obtained from this method.
The Oct4GiP reporter assay relies on efficient gene silencing. Effective siRNAs and efficient siRNA transfections are key to the success of the assay. Although we only described use of the reporter assay with siRNA transfections, the assay can also be performed with DNA transfections to silence or overexpress genes of interest. DNA transfection efficiency is usually lower than that of siRNAs and may reduce the strength of the phenotype.
Besides what was described in this protocol, the Oct4GiP reporter assay can be modified for identifying and characterizing genes that negatively regulate self-renewal as well. In that case, the cells can be transfected with siRNAs and cultured in differentiation conditions. Silencing of negative regulators of self-renewal will enhance self-renewal and sustain GFP expression, which can be detected by FACS similarly as described in the current protocol.
Besides the Oct4GiP reporter cells, other reporter cell lines using ES cell specific promoters, such as the Nanog-GFP 15-18 (Millipore, SCR089) and Rex1-GFP 19 cells, have also been generated. They can also be used to study ES cell self-renewal and maintenance using the same strategy. However, because there is noticeable difference in the activity and specificity of these ES cell marker gene promoters, these reporter cell lines behave differently in terms of background level and sensitivity and will thus complement each other. Finally, by using other lineage-specific reporter ES cell lines, our strategy can be modified to study fate-specification of ES cells and facilitate the use of ES cell as an in vitro model for mammalian early development.
No conflicts of interest declared.
We thank Brad Lackford for reading and editing the manuscript. This research was supported by the National Institute of Environmental Health Sciences, National Institutes of Health Intramural Research Program Z01ES102745 (to G. H.).
|ESGRO complete plus clonal grade medium||EMD Millipore||SF001-500P|
|DMEM (High glucose 1X)||Invitrogen||11965|
|OPTI-MEM(reduce serum medium)||Invitrogen||31985|
|ESGRO mLIF (107 units/1ml)||EMD Millipore||DAM1776540|
|MEM NEAA (Non-Essential Amino Acids)||Invitrogen||11140|
|100x Nucleosides for ES cell||EMD Millipore||10620-1|
|ES-qualified fetal bovine serum||Invitrogen||10437|
|Control siRNA: siRNA duplex targeting firefly luciferase (5’-CGTACGCGGAATACTTCGA) synthesized by Dharamcon.|