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Stable Expression of hrGFP by Mouse Embryonic Stem Cells: Promoter Activity in the Undifferentiated State and During Dopaminergic Neural Differentiation

Section on Development and Plasticity, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, National Institutes of Health, Department of Health and Human Services, Baltimore, Maryland, USA

Key Words. Embryonic stem cells • Promoter • hrGFP • Neural differentiation • Nestin • TH

Xianmin Zeng, Ph.D., Development and Plasticity Section, Cellular Neurobiology Research Branch, National Institute on Drug Abuse, 5500 Nathan Shock Drive, Baltimore, Maryland 21224, USA. Telephone: 410-550-6565 (ext 138); Fax: 410-550-1621; e-mail: xzeng{at}intra.nida.nih.gov

	ABSTRACT

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Three promoters, cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV), and Rous sarcoma virus (RSV) were examined for stable transgene expression in mouse embryonic stem (ES) cells and their progeny during dopaminergic neural differentiation. In undifferentiated ES cells the EF1 promoter was highly effective, while CMV had moderate activity. After 3 months in culture, expression of humanized renilla green fluorescent protein (hrGFP) was unchanged for the EF1 promoter and decreased for CMV. At the nestin-positive stage of differentiation, hrGFP and nestin were colocalized in about 20% of cells for EF1, in contrast to 80% of cells for the CMV promoter. In tyrosine hydroxylase (TH)-positive neurons neither the EF1 nor CMV promoter were effective. The RSV promoter was inactive in undifferentiated, nestin-positive, and TH-positive cells. Thus, EF1 and CMV are effective promoters for transgene expression in undifferentiated ES cells and nestin-positive neural precursors.

	INTRODUCTION

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Pluripotent mouse embryonic stem (ES) cells, derived from the inner cell mass of preimplantation embryos, are able to self-renew and have the capacity to generate any cell type of the developing embryo [1, 2]. ES cells have been used extensively to introduce genetic modifications into the mouse germline to create knockout mice [3]. ES cells also provide an important tool for the analysis of cell development in vitro, and genetic manipulation is critical for this purpose [4]. Many cellular and viral promoters have been successfully used for transgene expression [5–7], including cellular polypeptide chain elongation factor 1 alpha (EF1), cytomegalovirus (CMV) and Rous sarcoma virus (RSV), because of their strong activities in most cell lines. The RSV promoter is effective in several types of mammalian cells [8] and in some cells of the ventral mesencephalon [9], but inactive in ES cells [10]. In ES cells the EF1 promoter has been reported to be active in multiple stages of cell development, including naïve ES cells, embryoid bodies (EBs) and neuronal precursors [11]. The CMV promoter was reported to be inactive in undifferentiated ES cells by one group [11] and active by another group [12]. Both of these studies [11, 12] employed transient expression of the transgene.

The purpose of the present study is to examine stable expression of a transgene by several promoters in undifferentiated ES cells and during neural differentiation. Because of the interest in the development of dopaminergic neurons from ES cells, we employed a model in which ES cells are differentiated into dopaminergic neurons by coculture with the stromal cell line PA6 [13]. We chose two markers, nestin and tyrosine hydroxylase (TH) to monitor neural differentiation. Nestin is an intermediate filament protein expressed in mitotically active multipotent neural progenitors [14–18] including glia [19, 20] and certain other cell types such as myocytes and cardiomyocytes [17, 18]. TH is the rate-limiting enzyme in the biosynthesis of dopamine and is expressed in postmitotic dopaminergic neurons [21–23]. We established stable ES cell lines with the humanized renilla green fluorescent protein (hrGFP) under the control of the EF1, CMV, and RSV promoters and compared these promoters during the process of dopaminergic neurogenesis.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Vector Construction
pCMV/hrGFP was provided by Dr. Stephen Dewhurst (University of Rochester, Rochester, NY). For cloning pCMV/hrGFP, hrGFP was excised by BamHI and EcoRV from phrGFP (Stratagene; Cedar Creek, TX; http://www.stratagene.com.) and ligated into the BamHI and EcoRV sites of pcDNA3 (Invitrogen; Carlsbad, CA; http://www.invitrogen.com). For construction of pRSV/hrGFP, hrGFP was excised by NotI and EcoRV from phrGFP-1 (Stratagene) and ligated into the NotI and XbaI sites (both the insert and the vector were blunt before ligation) of pRC/RSV (Invitrogen). For cloning pEF1/hrGFP, the EF1 promoter was amplified by polymerase chain reaction (PCR) from pDrive-rEF/RU^TM (InvivoGen; San Diego, CA; http://www.invivogen.com) using primers with a BglII site at the 5' end and a NotI at the 3' end. The sequences of the primers were: 5'-CGGCAGATCTGGAGCCGAGAGTAA TTCATACAAAAG-3' and 5'-GCGCGCGGCCGCTGGC TTGGATCTGTAACGGCGCAG-3'. The PCR products were then digested with BglII and NotI, and ligated into the BglII and NotI digested followed by gel purification of pRSV/hrGFP backbone, so that the RSV promoter was replaced by the EF1 promoter. The promoters were sequenced and there were no mutations.

Cell Culture and Transfection
ES cells of the line 129/SVEV (Specialty Media; Phillipsburg, NJ; http://www.specialtymedia.com) were maintained on inactivated mouse embryonic fibroblast (MEF) feeder cells in Dulbecco’s modified Eagle’s medium supplemented with 15% fetal bovine serum, 2 mM non-essential amino acids, 2 mM L-glutamine, 50 µg/ml Penn-Strep (all from Invitrogen), 0.1 mM ß-mercaptoethanol (Specialty Media), and 1,000 U/ml recombinant murine leukemia inhibitory factor (ESGRO, Chemicon; Temecula, CA; http://www.chemicon.com). For electroporation, about 5 x 10⁶ cells in electroporation buffer (Specialty Media) were transfected with 20 µg of the desired reporter constructs using the BioRad Gene Pulser II (200 V, 500 µF) according to a standard protocol [24]. Neo^r clones were picked after 8–10 days of G418 selection and propagated using the same medium.

	NEURAL DIFFERENTIATION OF ES CELLS

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Neural differentiation of ES cells was induced by the mouse stromal cell line PA6 as described by Kawasaki et al. [13]. Briefly, transgenic ES cells were cultured to form colonies from a single cell on PA6 feeder cells in Glasgow minimum essential media (Invitrogen) supplemented with 10% knockout serum replacement (Invitrogen), 1 mM pyruvate (Sigma; St. Louis, MO; http://www.sigmaaldrich.com), 0.1 mM nonessential amino acids, and 0.1 mM ß-mercaptoethanol. ES cell colonies were grown at a density of 1,000 colonies per 3 cm dish. Medium was changed on days 4 and 6 and every day thereafter.

Immunocytochemistry and Colocalization
Nestin and TH expression were examined by immunocytochemistry following a standard protocol. In brief, ES cells grown on 24-well plates or 60 mm dishes were washed with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 10 min. The cells were washed with PBS and permeabilized with 0.1% Triton X-100 in PBS for 15 min. The cells were then rinsed with PBS, incubated with 0.5% SDS in PBS for 5 min, rinsed with PBS, and incubated in blocking buffer (5% goat serum, 0.1% Triton X-100 in PBS) for 1 hour. The cells were then exposed to either of the primary antibodies (1:200 dilution in 0.1% Triton X-100 + 2% goat serum in PBS): anti-nestin (Pharmingen; San Jose, CA; http://www.bdbiosciences.com/pharmingen), or anti-TH (Pelfreez; Rogers, AR; http://www.pelfreez-bio.com) at 4°C overnight. After washing with PBS, the cells were treated with secondary antibody (1:400 dilution in blocking buffer with 2% goat serum instead of 5% goat serum): either goat anti-mouse or goat anti-rabbit, Alexa fluor 594 (Molecular Probes; Eugene, OR; http://www.probes.com) for 1 hour. Finally the cells were washed with PBS and incubated with 30 µl of anti-fading solution, SlowFade Light (Molecular Probes). All steps were performed at room temperature unless specifically stated.

For the colocalization assay, cells growing on 12 mm or 22 mm coverslips were treated as described for immunocytochemistry. After incubation with the secondary antibody, the coverslips were mounted on Vectashield (Vector Laboratories; Burlingame, CA; http://www.vectorlabs.com) and examined with a laser scan confocal microscope (Zeiss 410). The red and green images were overlaid and analyzed with the Metamorp Imaging system 4.1 (Universal Imaging Corporation; Downingtown, PA; http://www.image1.com).

	RESULTS

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Establishment of Stable Cell Lines
A number of stable G418-resistant transgenic ES cell colonies were generated by electroporation of each of the pEF1/hrGFP, pCMV/hrGFP, and pRSV/hrGFP plasmids into ES cells. As seen in Figure 1, some G418-resistant ES colonies from transfection of the pEF1/hrGFP (Fig. 1A) and pCMV/hrGFP (Fig. 1B) constructs were hrGFP-positive. No hrGFP-positive colonies were found for the pRSV/hrGFP construct.

View larger version (31K): [in this window] [in a new window]   Figure 1. Promoter activity of EF1, CMV, and RSV in undifferentiated mouse ES cells. The ES cell line SVEV was transfected with each of the pEF1/hrGFP, pCMV/hrGFP, and pRSV/hrGFP plasmids by electroporation. Eight days after G418 selection, ES colonies were analyzed for hrGFP expression by fluorescence microscopy. A and C: pEF1/hrGFP, B and D: pCMV/hrGFP. Scale bar represents 20 µm.

For the CMVhrGFP/ES cells, expression of hrGFP was heterogeneous, as localized expression of hrGFP was found in most of the ES colonies (Fig. 1D). After colonies were dissociated, new hrGFP-negative colonies were formed from hrGFP negative cells. About half of the colonies were hrGFP-positive during the first several passages, and this percentage decreased with later passages. Only about 20% to 30% of colonies remained hrGFP-positive after 3 months of culture. No hrGFP-positive cells were found for the RSVhrGFP/ES cells.

The intensity of hrGFP was different for the undifferentiated EF1hrGFP/ES and CMVhrGFP/ES cells. The EF1hrGFP/ES cells were very strongly fluorescent, whereas only moderate fluorescence was observed for the CMVhrGFP/ES cells.

hrGFP Expression in Nestin-Positive Cells
Transgene expression was examined during differentiation of ES cells to nestin-expressing neural precursors, induced by growth on PA6 cells. Nestin was not expressed in undifferentiated ES cells, but started appearing at the edge of the differentiating ES colonies after 2 days of coculture with PA6 cells. At day 6, for both EF1hrGFP/ES (about 90% of the colonies were hrGFP-positive) and CMVhrGFP/ES (about 30% of the colonies were hrGFP-positive) cells, all the hrGFP-positive colonies also expressed nestin.

Confocal microscopy was used to examine the co-expression of nestin and hrGFP in individual cells. Only about 20% of the cells in hrGFP-positive colonies expressed both nestin and hrGFP for the EF1hrGFP/ES cells (Fig. 2A), despite expression of both hrGFP and nestin in the colony. In contrast, hrGFP and nestin were co-localized in about 80% of hrGFP-positive CMVhrGFP/ES cells (Fig. 2B). Furthermore, the expression of hrGFP by CMV became more homogeneous in the differentiating ES colonies (Fig. 2B), and the fluorescence signal was stronger as well.

View larger version (15K): [in this window] [in a new window]   Figure 2. Expression and co-localization of nestin and hrGFP from EF1hrGFP/ES, CMVhrGFP/ES, and RSVhrGFP/ES cell lines after a 5-day differentiation. More than 90% of the ES colonies from all three transgenic ES cell lines were nestin positive 5 days after neural induction by PA6. hrGFP expression in nestin-positive colonies from differentiating cells was similar to expression during propagation on mitotically inactive feeders for EF1hr GFP/ES cells (A). For CMVhrGFP/ES cells (B) hrGFP expression was almost homogeneous in differentiating cells, and not as patchy as in undifferentiated cells.

EF1 and CMV are Inactive in TH-Positive Cells
When ES cells were co-cultured with PA6 cells, TH-positive cells started appearing after 6 days and peaked at about day 14. By 14 days, about 90% of the EF1hr GFP/ES, CMVhrGFP/ES, and RSVhrGFP/ES colonies included TH-positive cells. Little or no hrGFP expression was seen in TH-positive neurons for either the EF1 or CMV promoter. Although most hrGFP-positive colonies contained TH-positive cells, the distribution of hrGFP and TH was found in different locations within the colonies. For both EF1hr GFP/ES and CMVhrGFP/ES cells, only a few cells (less than 1%) expressed both hrGFP and TH, while the majority of cells were positive for either hrGFP or TH, but not both (Fig. 3A and 3B). For the RSV promoter, there was no hrGFP expression in TH-positive neurons.

View larger version (29K): [in this window] [in a new window]   Figure 3. Expression and colocalization of TH and hrGFP from EF1hr GFP/ES, CMVhrGFP/ES, and RSVhr GFP/ES cell lines after 14-day neural differentiation. About 90% of the ES colonies from all the three transgenic ES cell lines were TH-positive after a 14-day coculture with PA6 cells. Although most hrGFP positive colonies contained TH-positive cells, few cells expressed both hrGFP and TH. A) EF1hrGFP/ES; B) CMVhrGFP/ES.

	DISCUSSION

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

Long-term expression of the hrGFP transgene in proliferating ES cells differed for the EF1 and CMV promoters. For the EF1 promoter, strong expression of hrGFP was present in more than 90% of ES cells, even after 3 months in culture. The expression of hrGFP by the CMV promoter gradually decreased, so that after 3 months only about 20% of CMVhrGFP/ES colonies remained hrGFP-positive. Also, for the EF1 promoter, in a clonal population of cells derived from an hrGFP-positive cell, the fluorescence produced by hrGFP was evenly distributed within the colony. In contrast, for the CMV promoter, individual colonies contained a mixture of hrGFP-positive and negative cells. Furthermore, EF1 produced stronger hrGFP expression than CMV in undifferentiated cells.

Differentiation of ES cells was monitored by nestin and TH expression. We did not observe nestin immunoreactivity in undifferentiated ES cells (data not shown), although expression of nestin in ES cells has been reported [28]. Nestin-positive colonies appeared in more than 90% of colonies within 5 days of coculture with PA6 cells. Colocalization of nestin and hrGFP was substantially less for the EF1 promoter as compared to the CMV promoter. Also, the CMV promoter produced a more consistent expression of hrGFP in nestin-positive ES colonies, unlike the heterogeneous expression seen in undifferentiated ES cells. The intensity of hrGFP fluorescence driven by CMV was also stronger in the differentiating ES cells, but still not as strong as for the EF1 promoter.

Transient expression of GFP in ES cells by several promoters including EF1 and CMV has previously been investigated [11, 12]. Chung et al. [11] reported robust activity of the EF1 promoter in undifferentiated ES cells, EBs, and neuronal precursors, whereas the CMV promoter was not active in undifferentiated ES cells or EBs, but was active in neuronal precursors. Ward and Stern [12] reported that the CMV promoter efficiently drove GFP expression in undifferentiated ES cells using five different ES cell lines. Our results are generally consistent with those of Chung et al. [11], except that CMV was not entirely inactive in undifferentiated ES cells in our experiments. Chung et al. used an IRES vector to express GFP by CMV, whereas we used a construct in which the hrGFP is directly driven by CMV similar to that used by Ward and Stern [11, 12]. The use of different vector constructs could explain some of the differences between these studies.

Neither the EF1 nor CMV promoter drove expression of hrGFP in TH-positive cells. After 6 days of co-culture with PA6, when TH-positive cells first appeared, hrGFP expression was still present for the EF1 and CMV promoters for most of the cells in hrGFP-positive colonies. As more cells developed the TH-positive phenotype (e.g., at day 14), hrGFP expression was lost. Thus both EF1 and CMV became inactive as the cells differentiated into mature neurons.

	CONCLUSION

Top Abstract Introduction Materials and Methods Neural Differentiation of ES... Results Discussion Conclusion References

 
Both EF1 and CMV were effective promoters in undifferentiated and partially differentiated ES cells. EF1 was most effective in driving hrGFP expression in undifferentiated ES cells, while CMV was more effective in neural precursors. None of the three promoters examined were effective in TH-positive neurons.

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