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. 2010 Sep 2;5(9):e12533.
doi: 10.1371/journal.pone.0012533.

NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

Yvonne Fischer  1 , Elvira Ganic, Jacqueline Ameri, Xiaojie Xian, Martina Johannesson, Henrik Semb
Affiliations

NANOG reporter cell lines generated by gene targeting in human embryonic stem cells

Yvonne Fischer et al. PLoS One. .

Abstract

Background: Pluripotency and self-renewal of human embryonic stem cells (hESCs) is mediated by a complex interplay between extra- and intracellular signaling pathways, which regulate the expression of pluripotency-specific transcription factors. The homeodomain transcription factor NANOG plays a central role in maintaining hESC pluripotency, but the precise role and regulation of NANOG are not well defined.

Methodology/principal findings: To facilitate the study of NANOG expression and regulation in viable hESC cultures, we generated fluorescent NANOG reporter cell lines by gene targeting in hESCs. In these reporter lines, the fluorescent reporter gene was co-expressed with endogenous NANOG and responded to experimental induction or repression of the NANOG promoter with appropriate changes in expression levels. Furthermore, NANOG reporter lines facilitated the separation of hESC populations based on NANOG expression levels and their subsequent characterization. Gene expression arrays on isolated hESC subpopulations revealed genes with differential expression in NANOG(high) and NANOG(low) hESCs, providing candidates for NANOG downstream targets hESCs.

Conclusion/significance: The newly derived NANOG reporter hESC lines present novel tools to visualize NANOG expression in viable hESCs. In future applications, these reporter lines can be used to elucidate the function and regulation of NANOG in pluripotent hESCs.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Gene targeting of NANOG in hESCs and expression of the eGFP reporter.
The strategy for gene targeting of the NANOG locus is presented schematically in A. The NANOG targeting vector was inserted into the 5′ untranslated region of the NANOG gene upstream of the NANOG start codon (ATG), resulting in an eGFP-tagged NANOG allele. Primer binding sites for PCR-based screening are indicated as arrows (A, lower panel). Red coloring marks targeting vector–derived NANOG sequences which replace endogenous NANOG sequences upon gene targeting. Abbreviations: bGl, rabbit beta-globin Intron 2; eGFP, enhanced green fluorescent protein gene; LP, locus of X-over P1 (recognition site for Cre-recombinase); NeoR, neomycin resistance gene; pA, polyadenylation site; Ex, exon; Ex1′, Ex2′, tuncated exon 1, 2. B) PCR experiments to screen for NANOG gene targeting events. A representative PCR screen of 3′ targeting events is shown in B, upper panel for three individual clones (lane 1 and 3: clones positive for 3′ gene targeting; lane 2: clone negative for 3′ gene targeting) with Neo/F and Ex3/R primers (fragment size 5,1kb). The NANOGeGFP BAC containing the reporter cassette integrated into the NANOG locus was used as positive control (BAC). m, DNA size marker. Correct targeting of the 5′ flanking region was tested by long-range PCR as shown in B, lower panel. PCR products of 13.3 kb were obtained with 5′/F and bGL/R primers with genomic DNA from NANeG1 and NANeG3 clones (lane 1, 2) and with the NANOGeGFP BAC template as positive control. C) The identity of the PCR products was verified by restriction enzyme digestion of the 3′PCR product with Hind III (upper panel) and SpeI/XhoI digestion of the 5′PCR product (lower panel). Expected fragments for 3′PCR products were 269bp, 965bp, 1.7kb and 2.2kb (marked by stars). Expected fragments for 5′PCR products were 374bp, 470bp, 1.7kb and 10.9kb (marked by stars). The 374bp and 470bp fragments are shown with higher magnification and contrast in the insert in C lower panel. Lanes contain PCR-products obtained with NANOGeGFP BAC (BAC), NANeG1 (1), NANeG3 (2), DNA size marker (m). XhoI (X) SpeI (S) and HindIII (H) sites within the gene targeted NANOG locus are indicated in A, lower panel. D) Histogram of eGFP expression levels in NANeG3 cells measured by flow cytometry. Wild-type HUES-3 cells were used as negative control. E) Brightfield (upper panel) and corresponding epifluorescence (middle) and merged (lower panel) images of NANeG1 and NANeG3 in a feeder-free culture system. Dashed lines indicate areas of spontaneous differentiation with loss of eGFP expression. Scale bar: 100µm.
Figure 2
Figure 2. Co-expression of NANOG and eGFP in NANeG lines.
Immunofluorescence staining of endogenous NANOG expression and co-localization with eGFP in NANeG lines growing on murine feeders (A) or in feeder-free culture (B). Arrows indicate areas of spontaneous differentiation with concomitant NANOG and eGFP downregulation. Scale bar: 100µm.
Figure 3
Figure 3. Modulation of reporter expression in NANeG lines.
A) Brightfield and corresponding epifluorescence images of NANeG1 undergoing differentiation as embryoid bodies are shown for day 2, 4 and 9 of differentiation. Scale bar: 100µm. Downregulation of eGFP expression during embryoid body differentiation was quantified by flow cytometry (B) and quantitative real-time PCR (C) in NANeG1 (upper panel) and NANeG3 (lower panel). D, E) NANeG cells growing in feeder-free culture were exposed to 0, 10, 50 or 100ng/ml Activin A (AA). Activin A induced eGFP expression in a dose-dependent manner, as shown by flow cytometry (D). Mean fluorescence intensities of the eGFP+ population (indicated in D) in NANeG3 (upper panel) and NANeG1 (lower panel) are shown in (E). For comparison, NANeG1 cells were grown on murine embryonic fibroblast feeders (MEF) without addition of Activin A.
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