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

Summary

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

Abstract

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.

Protocol

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.

1. Coat plates with 0.1% gelatin (Sigma) at room temperature for 30 minutes (0.1 ml gelatin solution/cm²).
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Discussion

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.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
ESGRO complete plus clonal grade medium	EMD Millipore	SF001-500P
DMEM (High glucose 1X)	Invitrogen	11965
0.25% Trypsin-EDTA	Invitrogen	25200
Lipofectamine 2000	Invitrogen	1001817
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
2-mercapt–thanol	Sigma-Aldrich	M7522-100ml
ES-qualified fetal bovine serum	Invitrogen	10437
Nanog siRNA	Invitrogen	MSS231181
Oct4 siRNA	Dharmacon	D-046256-02
Sox2 siRNA	Dharmacon	M-058489-01
Control siRNA: siRNA duplex targeting firefly luciferase (5’-CGTACGCGGAATACTTCGA) synthesized by Dharamcon.