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TECHNOLOGY DEVELOPMENT

Efficient Multicistronic Expression of a Transgene in Human Embryonic Stem Cells

^aLaboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan;
^bDepartment of Development and Differentiation, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto, Japan

Key Words. Human embryonic stem cells • Genetic modification • Multicistronic expression

Correspondence: Hirofumi Suemori, D.Sc., Laboratory of Embryonic Stem Cell Research, Stem Cell Research Center, Institute for Frontier Medical Sciences, Kyoto University, 53 Kawaharacho, Shogoin, Sakyo-ku, Kyoto 606-8507, Japan. Telephone: +81-75-751-3821; Fax: +81-75-751-3890; e-mail: hsuemori{at}frontier.kyoto-u.ac.jp

Received December 18, 2006; accepted for publication March 22, 2007.
First published online in STEM CELLS EXPRESS   March 29, 2007.

	ABSTRACT

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The applicability of human embryonic stem cells (hESCs) will be greatly enhanced by techniques that permit efficient genetic modification with multiple transgenes. We report here on single-promoter-driven foot-and-mouth disease virus segment 2A-mediated multicistronic expression of a transgene in hESCs. Efficient multicistronic expression of the transgene was permitted by 2A-mediated separation with almost the same amounts of encoded proteins in hESC. In addition, the multicistronic protein expression was successful in hESC-derived differentiated cells in in vivo and in vitro differentiation assays. This technology may be a significant advance in the genetic engineering of hESCs and hESC-derived cells for purposes that require the reliable expression of multiple transgenes.

Disclosure of potential conflicts of interest is found at the end of this article.

	INTRODUCTION

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Human embryonic stem cells (hESCs) may pave the way for applications in regenerative medicine and drug discovery [1, 2]. Before they can be used for such applications, it may be necessary to transfect them with multiple foreign genes to bestow desirable functions, including those for inducing cell differentiation, selecting the target differentiated cells, monitoring transplanted cells using marker genes, and generating disease model cells for drug discovery. Modifications can be effected using multiple transgenes by simultaneous transfection with different transgenes or by transfection with multiple expression cassettes on the same transgene. The former may result in contamination by single-transfected cells, and the latter may result in the suppression of transgene expression due to promoter interference [3]. A third alternative is the use of a transgene containing a single-promoter-driven multicistronic protein expression cassette. Such a multicistronic expression cassette would enable the simultaneous expression of multiple genes. The internal ribosomal entry site (IRES) is widely used to achieve multicistronic protein expression [4] (a schematic of IRES-mediated multicistronic expression is shown in Fig. 1A). Although it facilitates bicistronic protein expression when placed between adjacent genes in a single cassette, its primary disadvantage in human cells is poor downstream gene translation relative to the upstream gene [5]. An alternative to the IRES is multicistronic protein expression via the foot-and-mouth disease virus (FMDV) 2A segment [6, 7]. When several amino acids derived from the 2A segment are attached to the carboxyl end of the upstream protein and one amino acid is attached to the amino end of the downstream protein, 2A-mediated separation of the adjacent genes via a translational skip was reported to result in the multicistronic expression of the transgene in several mammalian cell types in vitro and in vivo [7–11] (schematic of 2A-mediated multicistronic expression is shown in Fig. 1B). However, no report exists that indicates that 2A-mediated multicistronic protein expression is efficient in a wide variety of human cell types, including hESCs.

View larger version (24K): [in this window] [in a new window]   Figure 1. Schematics of multicistronic protein expression from a transgene. (A): Schematic of multicistronic protein expression via IRES-mediated multiple translational initiation. (B): Schematic of multicistronic protein expression via the 2A-mediated translational skip mechanism. Abbreviations: IRES, internal ribosomal entry site; polyA, polyadenylation signal.

	MATERIALS AND METHODS

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Plasmid Construction
The 81-base pair 2A sequence of FMDV type O1K16, 5'-AAA ATT GTC GCT CCT GTC AAA CAA ACT CTT AAC TTT GAT TTA CTC AAA CTG GCT GGG GAT GTA GAA AGC AAT CCA GGT CCA-3' encoding amino acids KIVAPVKQTLNFDLLKLAGDVESNPGP, along with upstream BamHI, NruI, and PvuI restriction sites and downstream ScaI, SpeI, and partial AgeI restriction sites to facilitate cloning, was produced by annealing custom-designed complementary forward and reverse oligonucleotides. The plasmid pCMV-CFPnuc(2A)GFP was made by ligating the AseI-BamHI CMV-ECFP-nuc fragment from pECFP-nuc (Clontech, Palo Alto, CA, http://www.clontech.com) into AseI- and AgeI-digested pEGFP-N1 (Clontech) along with the annealed 2A oligonucleotides, which were constructed to maintain the same reading frame for both encoded proteins. The mutant 2A sequence (2Amut) was created by polymerase chain reaction and consisted of a single nucleotide, which mutated the terminal proline encoded by nucleotides CCA to GCA, which encodes an alanine; this product was cloned into the pGEM-T-Easy Vector (Promega, Madison, WI, http://www.promega.com), and the sequence was then examined. To construct pCMV-CFPnuc(2Amut)GFP, the subcloned 2Amut was digested as a BamHI-SpeI fragment from pGEM-T-Easy and then replaced into the original 2A sequence in BamHI- and SpeI-digested pCMV-CFPnuc(2A)GFP. Plasmid pCMV-GFP(2A)DsRed was constructed by ligating the AgeI-BamHI EGFP fragment from pEGFP-C1 (Clontech) and the NheI-NotI DsRed fragment from pDsRed-Express-N1 (Clontech) into the AgeI-BamHI and SpeI-NotI sites of pCMV-CFPnuc(2A)GFP, respectively. Plasmid pCMV-YFPmito(2A)CFPnuc(2A)DsRed was created by ligating the BamHI-NotI 2A-DsRed fragment and BsrGI-SpeI 2A fragment from pCMV-GFP(2A)DsRed with the NheI-BamHI ECFP-nuc fragment from pECFP-nuc into BsrGI- and NotI-digested pEYFP-mito (Clontech). Plasmid pCMV-CFPnuc(IRES)GFP was produced by ligating the AseI-MunI ECFP-nuc fragment of pECFP-nuc with AseI- and NheI-digested pIRES2-EGFP (Clontech). Plasmid pCMV-GFP(IRES)DsRed was made by ligating the EcoRI-NotI EGFP fragment from pEGFP-N1 and the SmaI-BstXI IRES fragment from pIRES2-EGFP into the EcoRI-SmaI site of pDsRed-Express-N1.

Plasmids with cytomegalovirus (CMV) promoters were used in the transient transfection experiments. For stable transfection, the CMV promoter was replaced with the CAG promoter to enhance the expression levels of each plasmid. To achieve this, the final products were digested with NotI and AseI, and the CMV promoter was replaced with the SalI-EcoRI CAG promoter fragment from the pCAGGS vector [12], and then the NotI-AflII SV40 polyA sequence was replaced with the EcoRI-HindIII globin polyA sequence from the pCAGGS vector.

hESC Culture and Transfection
The hESC lines KhES-1, -2, and -3 were maintained on a mouse embryonic fibroblast (MEF) feeder layer as previously described [13]. For fluorescence-activated cell sorting (FACS, see below) analysis or Western blotting, they were maintained in 35-mm dishes without a feeder-layer using MEF-conditioned medium [14]. To examine transgene expression, glass culture slides (BD Biosciences, San Diego, http://www.bdbiosciences.com) were used in place of plastic tissue culture dishes. The hESC cell lines were used in conformity with The Guidelines for Derivation and Utilization of Human Embryonic Stem Cells of the Ministry of Education, Culture, Sports, Science, and Technology, Japan.

For transient transfection, hESCs were transfected with 2 µg of DNA for 24 hours using FuGENE HD (Roche Diagnostics, Basel, Switzerland, http://www.roche-applied-science.com) according to the manufacturer's instructions and cultured in fresh medium for another 24 hours. The transgene expression of the transfected cells was then analyzed.

To isolate stable transfectants, cells were transfected with linearized DNA and selected by culturing on a neomycin-resistant MEF feeder in the presence of 200 µg/ml G418 disulfate salt solution (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) for 10–14 days. After selection, G418-resistant colonies were transferred to and maintained in separate new tissue culture plates, and their fluorescence protein expression was examined.

Examination of Transgene Expression
Cells were fixed with 4% paraformaldehyde (PFA) and stained with 4',6-diamino-2-phenylindole or propidium iodide to identify the nucleus. Fluorescence microscopy images were obtained using an Axio Imager Z1 (Carl Zeiss, Jena, Germany, http://www.zeiss.com) and analyzed using AxioVision software version 4.0 (Carl Zeiss).

For FACS analysis, the cells were washed twice with ice-cold phosphate-buffered saline (PBS) and then dissociated with 0.25% trypsin-EDTA into a single cell suspension for direct analysis. The proportions of cells expressing plasmid-induced green fluorescent protein (GFP) and DsRed fluorescence were examined using approximately 10,000 cells on a FACSCalibur flow cytometer (Becton, Dickinson and Company, Franklin Lakes, NJ, http://www.bd.com) with reference to a baseline of nontransfected hESCs.

For Western blotting, hESCs were dissociated with 0.25% trypsin-EDTA, washed several times with ice-cold PBS, and then lysed with sodium dodecyl sulfate-polyacrylamide gel electrophoresis loading buffer. For each sample, 5 µg of protein was then simultaneously separated on a 4%–20% linear gradient polyacrylamide gel, and the proteins were subsequently transferred to a polyvinylidene difluoride membrane (Bio-Rad, Hercules, CA, http://www.bio-rad.com). For primary antibodies, mouse monoclonal anti-GFP antibody (Ab-1; Lab Vision, Fremont, CA, http://www.labvision.com) was reacted against GFP and CFPnuc, and anti-DsRed antibody (Clontech) was reacted against DsRed. Anti-mouse horseradish peroxidase-conjugated IgG was then used as the secondary antibody. The protein bands were detected using Western Blotting Luminol Reagent (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com) and a Lumino Image Analyzer LAS-3000 (Fujifilm, Tokyo, http://www.fujifilm.com). Then, the expression level of each protein was quantified by Multi Gauge version 3.0 software (Fujifilm) using several gel images, which were obtained with different exposure times.

In Vivo and In Vitro Differentiation Assays
For embryoid body (EB) formation, confluent hESC colonies were detached, collected by sedimentation, and seeded onto low-attachment dishes (Costar Ultra Low Attachment; Corning Life Sciences, Acton, MA, http://www.corning.com/lifesciences). EBs were cultured for 2 weeks in the same medium as the hESCs and then plated onto gelatin- or fibronectin-coated culture slides (BD Biosciences) and cultured for another 2 weeks. The cells were next fixed with 4% PFA, incubated with anti-neural cell adhesion molecule (N-CAM) (Chemicon, Temecula, CA, http://www.chemicon.com) or anti-desmin (Sigma-Aldrich) polyclonal antibodies or anti--fetoprotein (AFP) monoclonal antibody (clone C3; Sigma-Aldrich) or anti-PDX-1 monoclonal antibody (clone 267712; R&D Systems) as the primary antibody and then detected using Alexa Fluor 546- or Alexa Fluor 350-conjugated secondary antibodies (Molecular Probes, Eugene, OR, http://probes.invitrogen.com) and fluorescence microscopy (Axio Imager Z1 and Axio Vision version 4.0).

For teratoma formation, hESCs were dissociated, and 200-µl suspensions containing approximately 1,000,000 cells were injected subcutaneously into severe combined immunodeficient mice (CLEA Japan Inc., Tokyo, http://www.clea-japan.com). After 8–12 weeks, the resulting teratomas were dissected and fixed with 4% PFA. Samples were embedded in O.C.T. compound (Sakura Finetechnical, Tokyo, http://www.sakura-finetek.com) sectioned at 7-µm thickness, and the sections were examined for fluorescence under fluorescent microscopy, whereas the adjacent sections were stained with hematoxylin and eosin.

	RESULTS

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To examine 2A-mediated bicistronic protein expression and its efficiency compared with IRES-mediated expression in hESCs, we constructed expression cassettes encoding the green fluorescent protein GFP and the red fluorescent protein from Discosoma (DsRed) separated by 2A or IRES, that is, GFP(2A)DsRed or GFP(IRES)DsRed, respectively (Fig. 2A), and transfected them into KhES-1 cells, a hESC line. Cells transfected with GFP(2A)DsRed showed GFP and DsRed fluorescence, whereas cells transfected with GFP(IRES)DsRed showed GFP fluorescence but no appreciable DsRed fluorescence (Fig. 2B). FACS analysis revealed that the population of dual fluorescence-positive cells among the GFP(2A)DsRed-transfected cells was distributed in a manner similar to the distribution in cells simultaneously transfected with GFP and DsRed, although the lower intensity of the fluorescence for both was slightly increased (Fig. 2C). Compared with cells transfected with GFP(2A)DsRed, only a few dual positive cells were detected among the GFP(IRES)DsRed-transfected cells, and most of them were distributed in the high-intensity GFP region.

View larger version (48K): [in this window] [in a new window]   Figure 2. Human embryonic stem cells (hESCs) and 2A-mediated multicistronic protein expression. (A): Schematic of the coding region of bicistronic constructs containing GFP and DsRed. (B): Fluorescent microscopic image of KhES-1 cells transfected with bicistronic constructs or nonbicistronic control constructs. The images used to examine fluorescence were obtained with the same exposure time for each filter set. (C): Fluorescence-activated cell sorting analysis of hESCs transfected with the bicistronic constructs or simultaneously transfected with nonbicistronic constructs (GFP+DsRed graph). The blue numbers indicate the percentages of their respective quadrants. (D): Schematic of bicistronic constructs containing CFPnuc and GFP. (E): Fluorescent microscopic image of KhES-1 cells transfected with the bicistronic constructs. The images were taken with the same exposure time under each microscopy filter, and weak optical leakage was observed between the GFP and CFP fluorescence filters. (F): Western blot analysis of bicistronic protein expression. (G): Schematic of the tricistronic constructs and fluorescent microscopic images of KhES-1, -2, and -3 cells. All scale bars indicate 10 µm. Abbreviations: CFP, cyan fluorescent protein; CFPnuc, CFP with nuclear localization signals; CMV, cytomegalovirus; DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein; IRES, internal ribosomal entry site; PI, propodium iodide; polyA, polyadenylation signal; YFP, yellow fluorescent protein.

To determine the separation efficiency of proteins flanking the 2A segment in hESCs, the CFPnuc and GFP expression levels were examined using Western blotting with a monoclonal antibody that recognizes both proteins (Fig. 2F). A 31-kDa band for CFPnuc or a 27-kDa band for GFP was observed in the control transfectant. In the CFPnuc(IRES)GFP transfectant, the 31-kDa CFPnuc protein band was observed together with the 27-kDa GFP protein band at an intensity ratio of 10:1. The CFPnuc(2A)GFP transgene produced a 27-kDa (2A)GFP band and a 34-kDa CFPnuc(2A) band that was a fusion between the 31-kDa CFPnuc protein and the upstream amino acids of 2A. The intensities of these two protein bands were similar (average 1:1.2) for the CFPnuc(2A)GFP transfectant. This indicates a 10-fold increase in GFP synthesis subsequent to 2A-mediated separation relative to IRES-mediated translation in hESC. In addition, a band possibly representing nonseparated CFPnuc and GFP was detected at 62-kDa in the Western blots. Comparison of the protein band intensities indicated a separation efficiency exceeding 93%. Similar separation efficiency was observed in a GFP(2A)DsRed transfectant (supplemental online Fig. 1). To show that the protein separation was due to 2A activity, a CFPnuc(2Amut)GFP transfectant was also examined. Analysis revealed a large amount of the 62-kDa band corresponding to the expected fusion protein along with weak single bands at 34 and 27 kDa, with a protein separation efficiency of less than 19%.

To investigate whether 2A-mediated separation is effective in the multicistronic expression of more than two genes, we constructed a multicistronic transgene encoding 2A, yellow fluorescent protein, with a mitochondrial targeting sequence (YFPmito), CFPnuc, and DsRed (Fig. 2E). In KhES-1 cells transfected with YFPmito(2A)CFPnuc(2A)DsRed, the proteins were successfully expressed via the tricistronic vector, and no adverse effects on localization were detected. This expression pattern indicates that 2A activity is not restricted in bicistronic constructs. Similar results were obtained using the hESC lines KhES-2 and KhES-3, showing that 2A-mediating separation is generally effective in hESC lines.

Next, we established transfected hESC lines with expression cassettes containing 2A to examine 2A-mediated bicistronic expression in stable transfectants. The 2A-mediated bicistronic expression was detected in the stably transfected hESCs (Fig. 3A). To investigate 2A-mediated bicistronic expression in all three germ layer cells derived from hESCs, in vitro differentiation was performed via EB formation using the CFPnuc(2A)GFP transformants. Fluorescence microscopy and immunostaining of N-CAM for neural cells (ectoderm), desmin for muscle cells (mesoderm), AFP for endodermal cells, and PDX-1 for pancreatic cells (definitive endoderm) revealed nuclear-localized CFPnuc and broad GFP expression in all three germ layers of the hESC-derived cells (Fig. 3B). We obtained similar results with in vitro EB formation and in vivo teratoma assay using GFP(2A)DsRed transformants (Fig. 2C–2E and supplemental online Fig. 2). In the teratomas, the GFP and DsRed expression levels differed among the tissues; for example, GFP expression was higher in neural cells, whereas DsRed expression was higher in muscle cells (Fig. 3E). This may have arisen from the differential degradation of each protein rather than different 2A-separating activity in each cell type, because GFP and DsRed expression was detected in all cells in the teratomas. Therefore, we concluded that 2A-mediated multicistronic expression is efficient in various hESC-derived cell types.

View larger version (99K): [in this window] [in a new window]   Figure 3. Human embryonic stem cell (hESC)-derived cells and 2A-mediated multicistronic protein expression. (A): Fluorescent microscopy and phase-construct image of an undifferentiated hESC transformant with bicistronic constructs. Scale bars, 100 µm. (B): Fluorescent microscopy and a phase-construct image of in vitro differentiated CFPnuc(2A)GFP transformants immunostained with anti-N-CAM, -desmin, -AFP, and -PDX-1 antibodies. Scale bars, 50 µm. (C): Fluorescent microscopy and bright-field image of a teratoma formed from a GFP(2A)DsRed transformant. (D): Fluorescent microscopic and HE-stained images of frozen sections of the teratoma. Scale bars, 1 mm. (E): High magnification images of neural and pigment epithelium, muscle, and intestine-like gut endoderm in the teratoma. Scale bars, 50 µm. Abbreviations: AFP, -fetoprotein; BF, bright field; CFP, cyan fluorescent protein; CFPnuc, CFP with nuclear localization signals; GFP, green fluorescent protein; HE, hematoxylin and eosin; N-CAM, neural cell adhesion molecule; polyA, polyadenylation signal.

	DISCUSSION

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When using the 2A segment for multicistronic protein expression, several 2A-derived amino acids remain attached to the carboxyl end of the upstream protein, and one amino acid remains attached to the amino end of the downstream protein. Therefore, functional examination of the transgene products, especially upstream proteins, might be necessary when using the 2A-segment. Indeed, it was reported that hrGFP-2A protein becomes more unstable than hrGFP protein in HEK293 cells [20]. We also found that DsRed-2A protein derived from the DsRed(2A)GFP construct did not display fluorescence in hESCs, even though the protein was detected by Western blotting (data not shown). However, the attachment of extra amino acids is a widely used method for tagging transgene products while leaving protein function intact (e.g., epitope tags such as the HA tag and Myc tag). Similar to tag fragments, the influence on protein functions by the 2A fragment has not been observed in most cases, including interleukin-12 [21], chloramphenicol acetyltransferase, ß-glucuronidase, puromycin N-acetyltransferase, HSV1-tk [22], alkaline phosphatase [23], superoxide dismutase 1 [24], HoxB4 [25], heparin-binding EGF-like growth factor [26], T-cell receptor [27], CD3 [8], CD8, p21 [9], cytochrome p450 [21], iduronidase, luciferase [10], and O-6-methylguanine-DNA-methyltransferase [11]. In addition, the upstream 2A-derived amino acids can be employed as a tag for immunoprecipitation assay through the use of anti-2A antibodies [28]. Although IRES-mediated multicistronic expression enables the production of untagged proteins, we showed that the IRES hardly works in hESCs, and that the 2A-mediated translational skip was superior in regard to multicistronic protein expression efficiency relative to IRES-mediated multiple translational initiation in hESCs.

The 2A-mediated multiple protein expression technique will provide an efficient and powerful tool for producing genetically modified hESCs for various purposes that require the introduction of multiple transgenes. The genetic alteration of hESCs is a very important and indispensable strategy for various research and applications such as regenerative medicine, drug screening, toxicogenomics, and functional genomics. Therefore, the 2A-mediated multicistronic expression could become an important technology in future hESC applications.

	DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST

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The authors indicate no potential conflicts of interest.

	ACKNOWLEDGMENTS

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This study was supported by grants from New Energy and Industrial Technology Development Organization (NEDO), Japan. K.H. and A.B.C. contributed equally to this work.

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