Transient and Stable Transgene Expression in Human Embryonic Stem Cells - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:53 |只看该作者 |倒序浏览 |打印

作者：Chee-Gee Liew, Jonathan S. Draper, James Walsh, Harry Moore, Peter W. Andrews作者单位：Centre for Stem Cell Biology, Department of Biomedical Science, Western Bank, University of Sheffield, Sheffield, United Kingdom 8 E1 d* J5 p% u






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      【摘要】
   Plasmid vectors remain a valuable yet capricious tool for the genetic manipulation of human embryonic stem (hES) cells. We have compared the efficacy of four promoters to mediate transient and stable transfection in hES and human embryonal carcinoma cell lines with the reporter enhanced green fluorescent protein (eGFP). In transient assays, the two mammalian promoters, UbiquitinC and Rosa26 (pUbiC and pR26), the human cytomegalovirus major immediate early promoter (HCMV-MIE; pCMV), and the HCMV-MIE/chicken ¦Â-actin/rabbit ¦Â-globin hybrid promoter (pCAGG) gave variable results that depended upon the cell line transfected but in an unpredictable way: each promoter supported strong transient expression in at least one cell line. The results for stable transfection were generally at variance with the transient assays. In each case, transgene silencing was quite marked, most notably with the pCMV, with which no eGFP-positive clones were obtained. An exception was the pCAG vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101; stable eGFP-positive transfectants were obtained, and these clones retained eGFP expression for over 120 passages, even in the absence of selection. However, if the PyF101 elements were removed, the resulting transfectants were also subjected to progressive gene silencing. Thus, the choice of promoter is critical for determining the desired effect of transgene expression in hES cells. Our data also demonstrate that pUbiC, pR26, pCAGG, and pCAG are more superior to the pCMV for generation of stable transfectants in hES cells.+ E% ~  P$ z3 ]' C
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Disclosure of potential conflicts of interest is found at the end of this article.
      【关键词】 Transfection Human embryonic stem cell Gene silencing Promoter& [) Q9 l6 }  v, A3 P
   INTRODUCTION2 m* b7 c7 ?1 W% e4 S
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Although a few reports have described the transfection of human embryonic stem (hES) cells, both in transient assays and for the generation of lines stably expressing specific transgenes  performed a series of transient transfection of hES cells with eGFP expression vectors driven by different promoter systems, but quantitative analysis of eGFP expression was not conducted. In this study, they found that they were unable to maintain stable expression of eGFP upon subsequent passage of undifferentiated hES cells.
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Against this background, we sought to compare the ability of several promoter systems to drive both transient and stable transgene expression in hES, and in hEC cells, their malignant counterparts from teratocarcinomas. We prepared four comparable vectors in which expression of eGFP is controlled by one of four commonly used promoters: the HCMV-MIEP (pCMV), either of two mammalian promoters, human UbiquitinC (pUbiC) . In transient assays, the individual promoter gave variable results that were cell line-dependent, and although for some promoters transgene silencing was common during stable clonal selection, the pCAGeGFP vector proved substantially resistant to this phenomenon. Several hES and hEC cell clones ubiquitously and constitutively expressing eGFP were derived. We showed that these stably transfected clones required the control of PyF101 enhancer in maintaining their transgene expression in the absence of puromycin selection.

MATERIALS AND METHODS
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Construction of the Plasmids6 l9 H: X4 a, l2 y3 K1 Q
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pCMV vector was constructed from peGFP-N1 (Clontech, Mountain View, CA, http://www.clontech.com) by inserting XhoI-NotI fragment containing the eGFP-1 from pd2eGFP-1 (Clontech) and ligated into the XhoI and NotI sites of peGFP-N1. For constructing pUbiC and pR26, the UbiC and R26 promoters were amplified from pUbiCeGFP-N1 and pR26eGFP-N1, respectively, by using oligonucleotide primers containing a BglII or SalI linker for each end. Polymerase chain reaction (PCR) products were then digested and ligated into the BglII and SalI sites of pd2eGFP-1. pUbiCeGFP-N1 plasmid is a gift from Peter Angel at German Cancer Research Center, Heidelberg, Germany, and pR26eGFP-N1 was kindly provided by Eric Sandgren, University of Wisconsin, Madison, WI. To construct pCAGeGFP, NotI- and XhoI-digested/Smad-interacting protein (SIP) fragments from the original pCAG-SIP plasmid (a gift from Austin Smith, Institute of Stem Cell Research, The University of Edinburgh, Edinburgh, U.K.) were excised and replaced with eGFP from pd2eGFP-1. pCAGG vector was constructed by subcloning CAGG promoter (digested with SalI and XhoI) into the pd2eGFP-1 vector. Finally, 1,165 bp of SalI (partial digestion) and XhoI fragment was excised from parental pCAGeGFP vector, resulting in pCAGPy. Primer sequences are available in online supplemental Table 1. See supplemental online Figure 1 for the plasmid maps used in this study.5 z7 R# W' o/ T
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Cell Culture/ H4 \; V6 C2 _* ~2 m! w
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hES and hEC cells were grown as previously described . In brief, hEC cells were maintained by growth in Dulbecco's modified Eagle's medium (DMEM; Invitrogen, Carlsbad, CA, http://www.invitrogen.com) supplemented with 10% fetal calf serum (FCS; Invitrogen) in a 37¡ãC humidified atmosphere of 10% CO2 in air. NTERA2 cells were passaged by scraping with a sterile 3-mm glass bead (Philip Harris Scientific, Leicestershire, U.K., http://www.philipharris.co.uk) and then seeding onto fresh tissue culture plastic. 2102Ep cells were passaged by treatment with 0.25% trypsin (w/v)/1 mM EDTA (Invitrogen), harvested, centrifuged, and then resuspended in DMEM/10% FCS onto fresh tissue culture plastic.

The hES cell lines H1, H7, and H14 were obtained from WiCell (WiCell Research Institute, Madison, WI, http://www.wicell.org). Shef3 hES cell line was derived from a preimplantation embryo donated for research by a couple attending an assisted conception unit within the U.K. and following fully informed patient consent complying with guidelines set out by the Human Fertilisation and Embryology Authority (HFEA) and NIH. The derivation process was permitted under HFEA license R0115 (H.M.). These hES cell lines were cultured in hES medium (knockout DMEM supplemented with 20% serum replacement, 1% nonessential amino acids, 1 mM L-glutamine ) under a humidified atmosphere of 5% CO2 in air at 37¡ãC. For subcultivation, the cells were harvested by treatment with 1 mg/ml collagenase type IV (Invitrogen) in DMEM/Ham's F-12 (Invitrogen) per T25 flask for 8¨C10 minutes at 37¡ãC, dispersed by scraping with a 3-mm glass bead, centrifuged at 68g for 3 minutes, and then seeded onto mouse embryonic fibroblast (MEF) feeders that had been washed once with phosphate-buffered saline (PBS; Invitrogen) immediately before use.6 g; d6 w  J7 F8 g( L. N. ]
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Transient and Stable Transfection Assay

Cells were seeded 1 day before transfection with the initial seeding density of 3 x 105 cells in six-well plates. Trypsin/EDTA (0.25%) was used to dislodge NTERA2 and 2102Ep hEC cells into a single cell suspension. For hES cells, cells were harvested from the dishes by 0.05% trypsin/EDTA treatment and then seeded on MEF feeders in hES medium for transient transfection or on matrigel-coated six-well plates and in MEF-conditioned medium for stable transfection assay (matrigel from BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Cells were approximately 70% confluent on the day of transfection. In preliminary experiments, plasmids were linearized before transfection (pR26 vector with SalI; pCAG with NotI), but we found no significant differences in transfection efficiencies compared with circular plasmids. Consequently, plasmids were not linearized in the experiments described here. Transfection was carried out with 9.5 µg of plasmid DNA using ExGen500 (MBI Fermentas, St. Leon-Rot, Germany, http://www.fermentas.com), SuperFect (Qiagen, Valencia, CA, http://www1.qiagen.com), and Lipofectin (Invitrogen) according to manufacturer's protocol. For electroporation, cell suspension were electroporated in small clumps at 240 V and 1,050 F with EquiBio electroporator (EquiBio, Maidstone, Kent, U.K., http://www.equibio.com). Detailed protocols of transfection and electroporation parameters are included in online supplemental Methods.# m8 a0 Y9 {& U! I" J
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For stable transfection assays, the cells were subjected to drug selection (400 µg/ml G-418 ) 24¨C48 hours after transfection. Distinct, antibiotic-resistant, individual colonies appeared after 2¨C3 weeks and were handpicked by micropipette, dissociated into small clumps of cells, and transferred into one well in a 12-well culture dish. The cells were then expanded in a six-well plate and continuously propagated in the presence of antibiotic. In some experiments, the cells were grown without antibiotic selection.: g) G6 B  p: d- a4 J

Flow Cytometry
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Cells were harvested by trypsinization and resuspended at 2 x 106 cells per ml in wash buffer (5% FCS) and 0.1% sodium azide (Sigma-Aldrich) in PBS. Antibodies were diluted in wash buffer (1:10 dilution), and 50 µl of each added per well of a 96-well plate. Cells 1 x 105 in a volume of 50 µl were added per well and then incubated for 30 minutes at 4¡ãC with gentle shaking. Plates were then spun at 280g for 3 minutes, and the supernatant discarded by inverting the plate. Cells were washed by adding 100 µl of wash buffer per well followed by centrifuging and discarding of the supernatant as previously described. Two further wash steps were performed, and then cells were resuspended in 50 µl of a 1:20 dilution of R-phycoerythrin-conjugated goat anti-mouse IgM and IgG secondary antibody (Dako Colorado, Ft. Collins, CO, http://www.dakousa.com) in wash buffer. The cells were incubated with the secondary antibody for 30 minutes at 4¡ãC with gentle shaking in the dark followed by three washes as above. Following the third wash, the cells were resuspended at 5 x 105 cells per ml in wash buffer and analyzed on a CyAn flow cytometer (Dako Colorado). In all cases, immunofluorescence with the specific antibodies was compared with that from a negative control antibody obtained from the parent myeloma cell line P3X63Ag8, as previously described .
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Neuronal Differentiation

NTERA2 cells were harvested as single cells, resuspended in DMEM/10% FCS, and seeded at 106 cells per 75-cm2 flask, in medium containing 10¨C5 M all-trans retinoic acid (Eastman Kodak, Rochester, NY, http://www.kodak.com). Pure neuronal cultures were obtained by a several rounds of differential replating as previously described  cells per 100-mm dish in DMEM/10% FCS supplemented with 1 pM cytosine arabinoside, 10 M fluorodeoxyuridine, and 10 M uridine (all from Sigma-Aldrich).
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For hES cells, H7 cells from confluent cultures were harvested and transferred to bacterial-grade Petri dishes. EBs formed after 1 week were transferred to a gelatin-coated tissue culture plate in media supplemented with 25 µg/ml insulin, 50 µg/ml transferrin, 30 nM sodium selenite, and 5 µg/ml fibronectin (all from Sigma). After 6 days, the cell outgrowths were transferred to a poly-D-lysine (Sigma-Aldrich)-coated tissue culture plate and grown in B27 (Invitrogen)/N2 (Invitrogen) media supplements containing 10 µg/ml laminin (BD Biosciences) and cultured for a further 6¨C15 days. ¦Â-Tubulin III expression of 4% paraformaldehyde (Sigma-Aldrich) in PBS-fixed neurons was visualized by immunostaining with a mouse anti-human TUJ-1 antibody (Covance Research Products, Berkeley, CA, http://www.covance.com) followed by anti-mouse Cy-3 (Sigma-Aldrich).$ p8 w, N5 ?7 F( t& |+ t( D' C
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Overexpression of PAX4 in hES Cells
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The pCAG-PAX4 expression vector was established by inserting a 1,161-kilobase human PAX4 coding sequence (CDS, NM_006193  ) into NotI and SalI restriction sites in place of eGFP in pCAG vector. Transfection and establishment of cell lines were carried out as above. Puromycin-resistant clones that appeared after 2 weeks of selection were picked and expanded in culture. After they were grown on a 100-mm dish for eight passages, cells were differentiated into EBs for 1 week and harvested in TriReagent (Sigma-Aldrich) for RNA isolation and reverse transcription-polymerase chain reaction (RT-PCR) analysis.3 Q! j3 R2 q9 R7 J8 H' n

Genomic DNA Analysis
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Genomic DNA (gDNA) was prepared using TriReagent. One microgram of gDNA was amplified for eGFP and ¦Â-actin. The cycle parameters were as follows: 95¡ãC for 1 minute, 58¡ãC for 1 minute, and 72¡ãC for 1 minute; 25 cycles. The PCR cycle was preceded by an initial denaturation of 3 minutes at 95¡ãC and followed by a final extension of 5 minutes at 72¡ãC. PCR products were separated by 1% agarose gel and visualized by UV transillumination (Syngene, Cambridge, U.K., http://www.syngene.com). Primer sequences are available in supplemental online Table 2.

Reverse Transcription-Polymerase Chain Reaction Analysis1 i* a0 n: ?& ?$ w: ]8 a1 E

Total RNA was extracted using TriReagent (Sigma-Aldrich) following manufacturer's instructions. The resulting total RNA was subjected to DNaseI treatment using DNA-free kit (Ambion, Austin, TX, http://www.ambion.com) at 37¡ãC for 30 minutes to remove any contaminating genomic DNA. Five micrograms of RNA was reverse transcribed using Moloney Murine Leukemia Virus reverse transcriptase and deoxynucleoside-5'-triphosphates (both from Promega). cDNA samples were then subjected to PCR amplification with human-specific primers. The cycle parameters were as follows: 95¡ãC for 1 minute, 52¡ãC or 58¡ãC for 90 seconds, and 72¡ãC for 1 minute; 30 cycles. Primer sequences and the size of the final products are described in supplemental online Table 2.- t! K2 ^3 G- f$ Y
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Statistical Analysis8 u1 e7 ^0 h& T* H- [1 W+ R6 D  j
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The data are presented as means ¡À standard error of three separate experiments. The significance of the differences in the transfection efficiencies using different protocols and differences in the promoter activity between different cell lines or the number of colonies formed was tested using t test analysis, with a p value

RESULTS
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Each Tested Promoter Exerts Transcriptional Activities in Transient Transfection9 W' K2 n" i; M9 u5 F

To investigate the transcriptional activities in hES and hEC cells, we prepared four comparable vectors in which eGFP expression is under the control of four different promoters: CMV, UbiC, R26, and CAGG (supplemental online Fig. 1). We first determined an optimal transfection method by comparing several commercially available cationic lipids and electroporation. Of the methods tested, ExGen500 produced the most eGFP-positive cells and highest level of eGFP expression in both hES and hEC cells (Fig. 1A and 1B); by propidium iodide staining, we estimated the viability of cells 24 hours after ExGen500 transfection was 80.56 ¡À 7.51% and 83.13 ¡À 6.69% for pCMV- and pCAGG-transfected cells, respectively (supplemental online Fig. 2).& r2 {2 e1 z* }  ]* l  t
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Figure 1. Transient transfection in hES and hEC cells. (A): hES and hEC cells were transfected by cytomegalovirus major immediate early promoter using several commercially available reagents and by electroporation. Representative fluorescence-activated cell sorting plots and histograms for transfection using Exgen500, Superfect, and Lipofectin are included for comparison. An empty vector (without eGFP) was used in transfection as control. The R1 gates define the eGFP-positive cells. (B): Transfection with ExGen500 seemed to deliver transgene much more efficiently into hES and hEC cells compared with SuperFect, Lipofectin, and electroporation. Experiments were done in triplicate. The error bars depict standard errors. The significance of differences in the transfection efficiencies was tested using t test analysis with a significance level of 5%. The asterisk (*) indicates that ExGen500-mediated transfection efficiency is significantly greater than those achieved using other reagents and electroporation (p

We next examined the transient transfection efficiencies in several hES and hEC cell lines transfected with different vectors using ExGen500 and analyzed the eGFP expression 24 hours after transfection (online supplemental Fig. 3). The percentage of transfected cells and the level of eGFP fluorescence were quantified by flow cytometry. Each of the vectors generated eGFP-positive cells, but there were significant differences between the cell lines and the vectors (Fig. 1C). In particular, H14 hES cells yielded significantly fewer eGFP-positive cells than the other lines with any of the vectors and almost none with the pUbiC vector. On the other hand, H1 cells yielded the most eGFP-positive cells when transfected by the pCMV, pR26, or pCAGG vectors, but almost none when transfected with the pUbiC vector. However, similar numbers of eGFP-positive Shef3 cells were produced by transfection with the pUbiC vector as by transfection with the other vectors, whereas in the two hEC cell lines, the pUbiC vector yielded the greatest number of transfectants. Further differences were evident when the expression level of the eGFP transgene was compared among the transfected cells. For example, although relatively few H14 transfectants were generated by the pCMV or pCAGG vectors, the mean fluorescence intensity of the eGFP-positive cells that were produced was comparable with that of eGFP-positive cells generated by the other vectors (Fig. 1D). Similar transient transfection efficiencies were observed when cells were transfected by pCAGG or pCAGeGFP vectors, since eGFP expression is driven by the same CAGG promoter (data not shown). Thus, the overall efficiency of transient transfection depended in an unpredictable fashion upon both the cell line and specific vector used.' H: x) g5 L  w. Z2 g$ M8 v

Expression of the pan-human antigen TRA-1-85/Ok(a)  (Fig. 2A and 2B). Thus, none of the vectors appeared to show any selective preference between the stem cells and their differentiated derivatives.( ^5 g% e% r$ n! |8 m+ |

Figure 2. Transient transfection and cell surface antigen expression of H7 cells analyzed by flow cytometry. Twenty four hours after transfection, cells were trypsinized, stained for cell surface markers with appropriate antibodies, and analyzed for eGFP and antigen expressions with a CyAn flow cytometer. (A): Flow cytometry scattered plot for SSEA1, SSEA3, or TRA-1-60 expression detected by R-phycoerythrin (fluorochrome, red; y-axis) and eGFP expression (FITC, green; x-axis) in pCMV-transiently transfected and untransfected/ unstained (control) H7 cells. (B): The table summarizes results of dual-color fluorescence-activated cell sorting analysis. eGFP and cell surface antigen expression of 2,000 cells were analyzed (shown here, H7 cells transfected by pCMV and pCAGG). The contingency table and 2 tests (degree of freedom, df = 1; significance level at 5%) revealed that transfection of eGFP driven by the cytomegalovirus promoter was not dependent on the expression of cell surface antigen (2  5). 2 5 was obtained in all different promoter-driven transfected human embryonic stem and human embryonal carcinoma cells. Abbreviations: eGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; pCMV, cytomegalovirus major immediate early promoter; SSEA, stage-specific embryonic antigen;  ve, positive; ¨Cve, negative.

Stable Transfection Assay# R8 r7 b# n' S; Q1 ^5 a# g# v8 S
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When stable transfectants were selected from two hEC cell lines (NTERA2 and 2102Ep) and one hES cell line (H7) that had each shown good susceptibility to transient transfection, no eGFP-positive colonies were found among the pCMV transfectants. However, stable drug-resistant colonies were found following transfection with pUbiC, pR26, pCAGG, and pCAGeGFP at an efficiency of 10¨C5. In the case of pUbiC, pR26, and pCAGG, only approximately 50% of the colonies were eGFP-positive (Fig. 3A). By contrast, all of the colonies obtained following transfection with pCAGeGFP expressed eGFP. In other experiments, pCAGeGFP was similarly effective in generating stable eGFP-expressing clones from another independently derived hES cell, Shef4 (data not shown).3 ?9 c: O" s6 C+ F, P% P

Figure 3. Stable transfection in human embryonic stem (hES) and human embryonal carcinoma (hEC) cells. (A): Two to 3 weeks after transfection, the number of colonies formed in a single well in six-well plates was scored. The bar graphs show the differences in the number of drug-resistant colonies and number of eGFP-positive colonies formed on a 9.5-cm2 culture area 3 weeks after transfection, during which antibiotic selection was applied. The data points represent the average number of colonies formed ¡ÀSEM, from three independent transfection experiments. t test analysis was used to analyze the differences in the number of drug-resistant or eGFP-positive colonies formed between different vectors with a significance level of 5%. *, indicates that the number of drug-resistant colonies formed following stable transfection with pCAGeGFP was significantly higher than that using other promoters in NTERA2 and 2102Ep cells. #, indicates that pCAGeGFP significantly generated more eGFP-positive colonies compared with other promoters in all cell lines. Several clones that were initially eGFP-positive were handpicked, expanded in culture, and subsequently grown in T25. (B): Representative graphs showing the proportion of eGFP-positive cells observed by flow cytometry in pUbiC, pR26, pCAGG, and pCAG-transfected NTERA2 cells and pUbiC and pCAG-transfected H7 cells on prolonged culture. Note the reduction in the number of eGFP-positive cells in all clones except for pCAG-derived clones (P, passage numbers). (C): Representative PCR analysis of genomic DNA extracted from transfected NTERA2 clones. The presence of eGFP transgene in eGFP-negative pCMV, pUbiC, and pR26-transfected cell population suggests that the loss of eGFP expression may be attributed to gene silencing. The eGFP sequence from plasmid DNA was used as positive control, and a sample containing no DNA (water only) was used as a negative control. (D): Fluorescence-activated cell sorting (FACS) analysis for the expression of eGFP after long-term passage in the absence of drug selection (Puromycin) in H7 hES cells. (E): FACS analysis for RA-treated NTERA2 cultures, which give rise to neurons. The R1 gates define the eGFP-positive cells. (F): These clones of which cells (H7 and NTERA2) maintain cell surface markers of undifferentiated hES cells, such as SSEA-3 and TRA-1-60, and differentiate into ¦Â-tubulin III-immunoreactive neuronal cells. (G): Removal of the PyF101 enhancer element from the pCAG vector resulted in its progressive silencing after prolonged passage without continued puromycin selection. Abbreviations: ¦Â-tub III, ¦Â-tubulin III; eGFP, enhanced green fluorescent protein; FITC, fluorescein isothiocyanate; P, passage number(s); pCMV, cytomegalovirus major immediate early promoter; pR26, Rosa26 promoter; pUbiC, UbiquitinC promoter; Puro, Puromycin; R, resistant; RA, retinoic acid; SSEA, stage-specific embryonic antigen; T25, 25-cm2 flask(s).# a/ b0 X1 j" a+ N) z. U" {

We then picked several eGFP-positive colonies from each transfection and expanded them in culture in the absence of antibiotic selection. In the case of the eGFP-positive pUbiC, pR26, and pCAGG transfectants, we observed a substantial loss of eGFP expression as the colonies were expanded, whereas the pCAGeGFP transfectants retained fully their eGFP expression, even though puromycin selection for the polycistronic message was no longer applied (Fig. 3B). Nevertheless, PCR analysis revealed the presence of eGFP DNA in all the eGFP-negative cells derived from the initially eGFP-expressing clones, implying that the loss of transgene expression in eGFP-negative cells might be attributed to progressive gene silencing, rather than loss of the transgene (Fig. 3C). The eGFP-positive transfectants of hES and hEC cells generated with the pCAGeGFP vector continued to retain robust eGFP expression for many passages in the absence of drug selection and during in vitro differentiation (Fig. 3D and 3E). Immunocytochemistry analysis revealed that the derived eGFP-positive clones maintained the expression of hES cell-specific antigens and eGFP was retained in their derivatives following differentiation, such as neurons (Fig. 3F). We have also derived eGFP-positive cytotrophoblast stem cell lines by co-culturing differentiated eGFP-positive EBs with endometrial stroma, confirming the unambiguous eGFP expression in undifferentiated and differentiated hES cells (data not shown, )., k: x- m4 _) y' f! g  u
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PyF101 Polyoma Enhancer Ensures Sustained Transgene Expression in pCAG-Transfected Clones in the Absence of Drug Selection

The lack of retention of eGFP expression in the pCAGG transfectants contrasted sharply with its retention in the pCAGeGFP transfectants, even though the same hybrid promoter is present in both vectors. However, the pCAGeGFP vector also included the PyF101 element from the polyoma enhancer, but this was absent from the pCAGG vector. To test whether the PyF101 element is responsible for the lack of silencing of the pCAGeGFP derived transfectants, we transfected NTERA2 cells with a derivative of the pCAGeGFP vector from which the PyF101 elements were removed (pCAGPy). Whereas these transfectants retained expression of eGFP when they were cultured with continued puromycin selection, a proportion of cells gradually lost eGFP expression over several passages in the absence of selection (Fig. 3G). Thus the PyF101 element seems to be responsible for the lack of gene silencing in the pCAGeGFP transfectants in contrast to the gene silencing seen in transfectants produced using the other vectors.) [# T' l% }/ M3 Z  z

Overexpression of PAX4 in hES Cells
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In vitro genetic manipulation of hES cells is likely to prove a valuable technology to research into the early stages of early development and knowledge of the genes involved in human embryogenesis. One such gene is the paired homeobox domain-4, PAX4, known to play a role in pancreatic ¦Â-cell development . To demonstrate the reliability of pCAG for the efficient and stable expression of genes other than eGFP, we overexpressed the human PAX4 gene CDS in H7 cells. Two weeks after transfection and continuous drug selection, we obtained approximately 15 puromycin-resistant colonies in a single well in a six-well plate at the efficiency comparable with that of cells transfected with pCAG-eGFP vector. These stable clones were expanded and maintained in the absence of puromycin selection. At passage 8, one of the PAX4-positive clones, PAX4 cl 5.8', was differentiated into EBs, collected at day 7, and assayed for PAX4 gene expression. We observed strong expression of the PAX4 CDS transgene in the undifferentiated PAX4-transfected H7 cells and in their EBs, confirming the constitutive transgene expression under the control of pCAG (Fig. 4). By contrast, RT-PCR analysis using a primer pair flanking the 5'-untranslated region of PAX4 revealed no expression of endogenous PAX4 in the untransfected H7 cells but upregulation expression during in vitro differentiation in the PAX4-positive EBs. This pattern of expression was not seen in untransfected H7 cells.+ J" y1 n% e& p$ m- E7 x; O6 B8 F

Figure 4. Overexpression of human PAX4 CDS in H7 cells. Reverse transcription-polymerase chain reaction analysis of transfected PAX4 CDS and endogenous PAX4 UTR expression in untransfected parental H7 and PAX4-positive H7 undifferentiated (ES) and differentiated (EBs) samples. PAX4 CDS was expressed in undifferentiated and differentiated PAX4-transfected H7 cells, but the endogenous PAX4 gene, detected by primer sequence flanking 5'-untranslated region of PAX4 CDS, was expressed only in PAX4-positive EBs. There was no PAX4 expression in untransfected parental cells. Sample containing no cDNA (water only) was used as negative control. Abbreviations: CDS, coding sequence; ES, embryonic stem; UTR, untranslated region.

DISCUSSION
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Transient and stable transfection provides a powerful tool to establish the function of proteins as well as the effect of mutations on gene function in hES cells. In this study, we have compared the transcriptional activity and strength of several commonly used promoters. Plasmid vectors were used for this purpose since they potentially provide a rapid screening tool for various transcription factors that may function in hES cells. Although lentiviral infection is proven to produce a high proportion of stable integrants in hES cells .

We demonstrated that hES and hEC cells can be efficiently transfected to express a gene of interest. Flow cytometry and UV microscopy revealed that the overall transient transfection efficiency depended upon the cell line and specific vectors used. The CMV, R26, and CAGG promoters all demonstrated robust activities in hES and hEC cell-transient transfection. However, the UbiC promoter activity varied noticeably, and only low eGFP expression was observed following transfection into H1 and H14 hES cells, whereas strong expression from UbiC was seen in the hEC cells. Compared with hEC cells, a more distinct difference in individual promoter activity was observed between hES cell lines, suggesting that hES cell lines may vary considerably in terms of the expression of relevant transcription factors. It also seems that none of the promoters demonstrated any selective preference in transfection of either undifferentiated or spontaneously differentiated cells in the hES cultures; the transfection efficiencies in undifferentiated SSEA3( ), TRA-1-60( ), or SSEA1(¨C) stem cell populations were similar to that in the bulk culture.
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A lot of transgene expression studies involve the study of a gene over a longer period of time. In such an approach, constitutive transgene expression in stable transfection assay would require an efficient promoter expression vector that resists gene silencing. In a previous study, Kim et al.  found that very few drug-selectable transfectants could be obtained from undifferentiated hES cells and few, if any, stably maintained the expression of an eGFP transgene upon subculture, although they reported that hEC cells do appear to maintain stable transgene expression over several passages. Here, however, we found that we could obtain stable transfectants of both hES and hEC cells using pUbiC, pR26, pCAGG, and pCAGeGFP, although not with pCMV.

In the subsequent study, we expanded and maintained these eGFP-expressing clones in culture. There was a reduction in the number of eGFP-expressing cells in some of the stably transfected clones following transfection with pUbiC, pR26, and pCAGG vectors, indicating that gene silencing is not an infrequent outcome following hES cell transfection. We also detected the same pattern of loss of eGFP expression in our hEC cell lines, suggesting the close similarity between these two cell types. On the other hand, using the pCAGeGFP vector, in which the CAGG composite promoter is linked to the polyoma virus mutant enhancer PyF101 and an IRES, we generated several eGFP-expressing hES and hEC cell lines that maintained their eGFP expression ubiquitously and constitutively even after antibiotic selection was subsequently removed. We have maintained these cells in culture for more than 120 passages. Using the CAGG-IRES vector, Vallier et al.  have also derived fluorescent hES cell lines. However, we further demonstrated that retention of eGFP expression in clones generated using the pCAGeGFP vector is attributed to the activity of polyoma virus PyF101 mutant enhancer. Thus, the choice of promoter and expression cassette in the transfection are critical factors to consider for establishing stable hES cell lines.7 v9 A( y  ]; V8 s0 U. ~) X

Our results indicate that gene silencing is a significant problem for stable transfection of hES and hEC cells using many promoter systems, and could therefore interfere with efforts to produce conditional expression systems in these cells. However, the CAGG promoter in the pCAG vector containing PyF101 elements provides a robust system for deriving long-term constitutive transfectants, not only for reporter such as eGFP, but also for key developmental genes such as PAX4. Genetic manipulation of hES and hEC cells is likely to provide a useful model for using these cells to study self-renewal, human embryogenesis, and migration of differentiated cells in transplantation studies.
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DISCLOSURES OF POTENTIAL CONFLICTS OF INTEREST
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The authors indicate no potential conflicts of interest.

ACKNOWLEDGMENTS
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We thank Christine Pigott for cell culture, Mark Jones, Ian Morton for flow cytometry analysis, and Gaetano Zafarana and Paul Gokhale for helpful discussion. This work was supported by grants from the Juvenile Diabetes Research Foundation, Biotechnology and Biological Sciences Research Council, and Medical Research Council. C.-G.L. is currently affiliated with the University of California Irvine Stem Cell Research Center, University of California, Irvine, CA.  Q( c7 [# K' H- A  u7 f+ a
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