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.).

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 &mu;M &b...

<ol>
	<li>Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+stem+cell+differentiation:+emergence+of+a+new+era+in+biology+and+medicine.">Embryonic stem cell differentiation: emergence of a new era in biology and medicine.</a> Genes Dev. 19, 1129-1155 (2005).</li><li>Murry, C. E., Keller, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differentiation+of+embryonic+stem+cells+to+clinically+relevant+populations:+lessons+from+embryonic+development.">Differentiation of embryonic stem cells to clinically relevant populations: lessons from embryonic development.</a> Cell. 132, 661-680 (2008).</li><li>Hu, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-wide+RNAi+screen+identifies+a+new+transcriptional+module+required+for+self-renewal.">A genome-wide RNAi screen identifies a new transcriptional module required for self-renewal.</a> Genes Dev. 23, 837-848 (2009).</li><li>Ying, Q. L., Nichols, J., Evans, E. P., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Changing+potency+by+spontaneous+fusion.">Changing potency by spontaneous fusion.</a> Nature. 416, 545-548 (2002).</li><li>Ying, Q. L., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Defined+conditions+for+neural+commitment+and+differentiation.">Defined conditions for neural commitment and differentiation.</a> Methods Enzymol. 365, 327-341 (2003).</li><li>Nichols, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+pluripotent+stem+cells+in+the+mammalian+embryo+depends+on+the+POU+transcription+factor+Oct4.">Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4.</a> Cell. 95, 379-391 (1998).</li><li>Niwa, H., Miyazaki, J., Smith, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+expression+of+Oct-3/4+defines+differentiation,+dedifferentiation+or+self-renewal+of+ES+cells.">Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells.</a> Nat. Genet. 24, 372-376 (2000).</li><li>Ding, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+genome-scale+RNAi+screen+for+Oct4+modulators+defines+a+role+of+the+Paf1+complex+for+embryonic+stem+cell+identity.">A genome-scale RNAi screen for Oct4 modulators defines a role of the Paf1 complex for embryonic stem cell identity.</a> Cell Stem Cell. 4, 403-415 (2009).</li><li>Chambers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+expression+cloning+of+Nanog,+a+pluripotency+sustaining+factor+in+embryonic+stem+cells.">Functional expression cloning of Nanog, a pluripotency sustaining factor in embryonic stem cells.</a> Cell. 113, 643-655 (2003).</li><li>Masui, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pluripotency+governed+by+Sox2+via+regulation+of+Oct3/4+expression+in+mouse+embryonic+stem+cells.">Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells.</a> Nat. Cell Biol. 9, 625-635 (2007).</li><li>Mitsui, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+homeoprotein+Nanog+is+required+for+maintenance+of+pluripotency+in+mouse+epiblast+and+ES+cells.">The homeoprotein Nanog is required for maintenance of pluripotency in mouse epiblast and ES cells.</a> Cell. 113, 631-642 (2003).</li><li>Fazzio, T. G., Huff, J. T., Panning, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+RNAi+screen+of+chromatin+proteins+identifies+Tip60-p400+as+a+regulator+of+embryonic+stem+cell+identity.">An RNAi screen of chromatin proteins identifies Tip60-p400 as a regulator of embryonic stem cell identity.</a> Cell. 134, 162-174 (2008).</li><li>Pease, S., Braghetta, P., Gearing, D., Grail, D., Williams, R. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Isolation+of+embryonic+stem+(ES)+cells+in+media+supplemented+with+recombinant+leukemia+inhibitory+factor.">Isolation of embryonic stem (ES) cells in media supplemented with recombinant leukemia inhibitory factor.</a> 141, 344-352 (1990).</li><li>Young, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+the+embryonic+stem+cell+state.">Control of the embryonic stem cell state.</a> Cell. 144, 940-954 (2011).</li><li>Chambers, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanog+safeguards+pluripotency+and+mediates+germline+development.">Nanog safeguards pluripotency and mediates germline development.</a> Nature. 450, 1230-1234 (2007).</li><li>Maherali, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Directly+reprogrammed+fibroblasts+show+global+epigenetic+remodeling+and+widespread+tissue+contribution.">Directly reprogrammed fibroblasts show global epigenetic remodeling and widespread tissue contribution.</a> Cell Stem Cell. 1, 55-70 (2007).</li><li>Rodda, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transcriptional+regulation+of+nanog+by+OCT4+and+SOX2.">Transcriptional regulation of nanog by OCT4 and SOX2.</a> J. Biol. Chem. 280, 24731-24737 (2005).</li><li>Schaniel, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+of+short+hairpin+RNAs--triggers+of+gene+silencing--into+mouse+embryonic+stem+cells.">Delivery of short hairpin RNAs--triggers of gene silencing--into mouse embryonic stem cells.</a> Nat. Methods. 3, 397-400 (2006).</li><li>Toyooka, Y., Shimosato, D., Murakami, K., Takahashi, K., Niwa, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+and+characterization+of+subpopulations+in+undifferentiated+ES+cell+culture.">Identification and characterization of subpopulations in undifferentiated ES cell culture.</a> Development. 135, 909-918 (2008).</li></ol>

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 scr...

<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>ESGRO complete plus clonal grade medium</td>
			<td>Millipore</td>
			<td>SF001-500P</td>
		</tr>
		<tr>
			<td>DMEM (High glucose 1X)</td>
			<td>Invitrogen</td>
			<td>11965</td>
		</tr>
		<tr>
			<td>0.25% Trypsin-EDTA</td>
			<td>Invitrogen</td>
			<td>25200</td>
		</tr>
		<tr>
			<td>Lipofectamine 2000</td>
			<td>Invitrogen</td>
			<td>1001817</td>
		</tr>
		<tr>
			<td>OPTI-MEM(reduce serum medium)</td>
			<td>Invitrogen</td>
			<td>31985</td>
		</tr>
		<tr>
			<td>ESGRO mLIF (107 units/1ml)</td>
			<td>Millipore</td>
			<td>DAM1776540</td>
		</tr>
		<tr>
			<td>MEM NEAA (Non-Essential Amino Acids)</td>
			<td>Invitrogen</td>
			<td>11140</td>
		</tr>
		<tr>
			<td>100x Nucleosides for ES cell</td>
			<td>Millipore</td>
			<td>10620-1</td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Sigma</td>
			<td>M7522-100ml</td>
		</tr>
		<tr>
			<td>ES-qualified fetal bovine serum</td>
			<td>Invitrogen</td>
			<td>10437</td>
		</tr>
		<tr>
			<td>Nanog siRNA</td>
			<td>Invitrogen</td>
			<td>MSS231181</td>
		</tr>
		<tr>
			<td>Oct4 siRNA</td>
			<td>Dharmacon</td>
			<td>D-046256-02</td>
		</tr>
		<tr>
			<td>Sox2 siRNA</td>
			<td>Dharmacon</td>
			<td>M-058489-01</td>
		</tr>
	</tbody>
</table>
Control siRNA: siRNA duplex targeting firefly luciferase (5'-CGTACGCGGAATACTTCGA) synthesized by Dharamcon.

oct4gip reporter assay to study genes that regulate mouse embryonic stem cell maintenance and self-renewal

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.

Watch this Scientific Journal Video about Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal at JoVE.com

Oct4GiP Reporter Assay to Study Genes that Regulate Mouse Embryonic Stem Cell Maintenance and Self-renewal

We describe a fluorescence reporter assay to quickly identify and characterize genes that regulate mouse embryonic stem cell maintenance and self-renewal.

Pluripotency and self-renewal are two defining characteristics of embryonic stem cells (ES cells). Understanding the underlying molecular mechanism will ...

oct4gip-reporter-assay-to-study-genes-that-regulate-mouse-embryonic

Research

JoVE Journal

Biology

10.4K visualizações.  National Institute of Environmental Health Sciences. Nós descrevemos um ensaio repórter de fluorescência para rapidamente identificar e caracterizar genes que regulam a manutenção do mouse células-tronco embrionárias e auto-renovação.