First published online June 7, 2005
Stem Cells
Vol. 23 No.
8
September 2005, pp.
1028
-1034
doi:10.1634/stemcells.2004-0335; www.StemCells.com
© 2005 AlphaMed Press
Stable and Uniform Gene Suppression by Site-Specific Integration of siRNA Expression Cassette in Murine Embryonic Stem Cells
Guo Dong Zhenga,
Kyoko Hidakaa,
Takayuki Morisakia,b
a Department of Bioscience, National Cardiovascular Center Research Institute;
b Department of Molecular Pathophysiology, Osaka University Graduate School of Pharmaceutical Sciences, Suita, Osaka, Japan
Key Words. Embryonic stem cells • Green fluorescent protein • Small interfering RNA • Homologous recombination • Hprt locus • Flow cytometry
Correspondence: Takayuki Morisaki, M.D., Ph.D., Department of Bioscience, National Cardiovascular Center Research Institute, 5-7-1 Fujishirodai, Suita, Osaka 565-8565, Japan. Telephone: 81-6-6833-5012, ext. 2506; Fax: 81-6-6835-5451; e-mail: morisaki{at}ri.ncvc.go.jp
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ABSTRACT
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We developed a simple system to introduce small interfering RNA (siRNA) into murine embryonic stem cells (ESCs) and then showed its stable and uniform expression. Using hypoxanthine guanine phosphoribosyl transferase 1 (Hprt)–deficient ESCs as a recipient, we efficiently introduced an siRNA expression cassette into the Hprt locus by homologous recombination, which was easily detected by HAT selection. Nearly all of the HAT-resistant clones exhibited a silenced expression of the exogenous target gene (enhanced green fluorescent protein [EGFP]) or the endogenous target gene (Flk1). Flow cytometry profiles demonstrated that there were no significant differences in level of suppression among individual clones and cells. The suppressing effect by siRNA was maintained for more than 1 month in both undifferentiated and differentiated ESCs, while its persistent expression did not disturb their growth or differentiation potential. The stable and uniform suppression capability of this system will help to screen genes and provide important information regarding cell differentiation in ESCs.
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INTRODUCTION
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Embryonic stem cells (ESCs) are pluripotent cell lines derived from the inner cell mass of blastocyst-stage embryos that have been shown to differentiate in vivo and in vitro into all cell lineages of adult animals. Although there is considerable interest in the therapeutic applications of ESCs [1, 2], they cannot be used in regenerative therapies in an undifferentiated state, because they form teratomas after transplantation [3]. Hence, a thorough understanding of the process of lineage commitment and differentiation of ESCs is essential for developing transplantation strategies [4].
To understand the specific signaling pathways involved in specification and differentiation of ESC-derived cells, it is important to elucidate the function of specific genes by manipulating their level of expression [5]. In mouse studies, homologous recombination–based gene knockout methods have been shown to be powerful tools to understand the function of genes, and several reports have demonstrated that knockout of developmentally important genes has an effect on differentiation ability in vitro [6, 7]. However, knockout methods are used infrequently in in vitro studies because an additional round of transfection is required to knock out all of the alleles.
RNA interference (RNAi) is a recently elucidated technique that has been used successfully to suppress gene expression in Caenorhabiditis elegans and Drosophila melanogaster. In mammalian cells, including mouse and human ESCs, it was recently shown that small interfering RNA (siRNA), administered as a 21-nt double-stranded RNA, could suppress gene expression, though the knockdown effect was transient and could be maintained only for a short term [8, 9]. Very recently, a hairpin siRNA expression system driven by the RNA polymerase III promoter was shown to be able to suppress target gene expression in ESCs [5, 10–13]. This vector-based technology is a simple and promising means for exploring the biological functions of differentiation-related genes in ESCs, whereas the availability of an RNAi technique for ESCs also provides a convenient method for directly generating knockdown mice, by utilizing tetraploid embryo [14–16]. In the present study, we found that an siRNA expression cassette, site-specifically integrated into the constitutively active hypoxanthine guanine phosphoribosyl transferase 1 (Hprt) locus, uniformly suppressed target gene expression over a long term. As speculated, the effect of gene suppression was highly reproducible and persisted throughout the process of differentiation by ESCs.
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MATERIALS AND METHODS
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ESC Culture and Transfection
CGR8 ESC-derived murine ESC lines, ht7 and its derivatives, were maintained in the absence of feeder cells in Glasgow’s modified Eagle’s medium (GMEM) supplemented with 1% fetal calf serum (FCS), 10% Knockout Serum Replacement (Invitrogen, Carlsbad, CA, http://www.invitrogen.com), 2-mercaptoethanol, and leukemia inhibitory factor, as previously described [17]. The culture medium used for maintenance also contained 100 µg/ml of hygromycin to select Oct4-positive stem cells, because these ESCs carry the hygromycin phosphotransferase gene at the Pou5f1 (Oct4) locus [18, 19]. Differentiation of ESCs was induced by embryoid body (EB) formation, as described previously [17, 20]. Briefly, 500 cells were cultured in 20 µl of differentiation medium without leukemia inhibitory factor in a hanging drop system for 2 days to generate cell aggregates, then further incubated in bacteriological dishes as floating EBs for 5–7 days. To observe cell differentiation, EBs were transferred into gelatin-coated 24-well dishes on day 5. Typically, spontaneous beating was first observed on day 7 or 8, and more than 90% of the EBs contained beating foci on day 8 or 9. For stable transfection with targeting vectors, 1 x 107 cells were plated onto a 10-cm gelatin-coated dish and transfected with 10 µg of linearized targeting vectors using Lipofectamine 2000 (Invitrogen). The next day, the cells were transferred to three 10-cm plates and 2 days after transfection, drugs were added for selection of drug-resistant cells. Drug-resistant colonies that appeared within 8–10 days after transfection were selected, then dissociated and plated onto 24-well gelatin-coated plates, and used for further analysis.
Construction of Hprt-Deficient Founder ESCs with or Without Constitutive Expression of the Enhanced Green Fluorescent Protein Gene
Because ht7 ESCs are Hprt positive (Hprt+), the first exon of the Hprt gene was deleted and replaced with the neomycin phosphotransferase–expressing cassette (MC1neo), using an Hprt-deleting vector derived from p
Hprt [15]. After preselection with G418 (EMD Biosciences, San Diego, http://www.emdbiosciences.com) at 400 µg/ml, 6-thioguanine (6-TG)–resistant cells were selected as Hprt-deficient cells. The integrity of the targeting event was confirmed by Southern blotting, and a single cell line, termed hdh31, was used for introduction of siRNA expression cassettes for endogenous target genes. As an exogenous target gene, the enhanced green fluorescent protein (EGFP) gene was introduced into the Gt(ROSA)26Sor gene trap locus(ROSA26)using the ROSA26 targeting vector, pROSA26-1. Constitutive expression of the EGFP gene was driven by the CAG promoter (cytomegalovirus [CMV] enhancer/chick ß-actin promoter derived from pCX-EGFP [21]). The resultant Hprt–EGFP+ cell line was named hgh2 and used to monitor the effect of siRNA for EGFP. The cell lines used in this study are summarized in Table 1
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Construction of pHprt-siGFP and pHprt-siFlk1 Plasmids
An Hprt targeting vector containing the first and second exons of the Hprt gene was derived from pMP8NEBlacZ [15]. To construct an siRNA-expressing cassette for GFP (hereafter termed siGFP), the sequence containing the human H1 promoter and short hairpin small interfering sequence for GFP, derived from pGtoR [22], was amplified by polymerase chain reaction (PCR) and cloned into the Hprt targeting vector to generate pHprt-siGFP. To construct siRNA-expressing cassettes for the Flk1 gene (termed siFlk1), the sequence containing the H1 promoter was first cloned into the Not I-Xho I site of the Hprt targeting vector to generate pHprt-H1. Three different short hairpin small interfering sequences for the Flk1 gene (siFlk1-A, -B, and -C) were then directly cloned into the Bam HI -Xho I site of pHprt-H1 to generate pHprt-siFlk1A, -siFlk1B, and -siFlk1C. The loop sequence was derived from human microRNA, miR-23 [23]. The oligos used for cloning were as follow: siFlk1-A top: 5'- GATCCGAGCG GAGACGCTCT TCATAAGCTT CCTGTCACTT ATGAAGAGCG TCTCCGCTCT TTTTTggaaaC -3', siFlk1-A bottom: 5'- tcgaGtttcc AAAAAAGAGC GGAGACGCTC TTCATAAGTG ACAGGAAGCT TATGAAGAGC GTCTCCGCTCG -3', siFlk1-B top: 5'- GATCCGGACG GATGATCAAG AGAAATGCTT CCTGTCACAT TTCTCTTGAT CATCC GTCCT TTTTTggaaaC -3', siFlk1-B bottom: 5'- tcgaGtttcc AAAAAAGGAC GGATGATCAA GAGAAATGTG ACAGGAAGCA TTTCTCTTGA TCATCCGTCCG -3', siFlk1-C top: 5'- GATCCGGAAG TGACTGAAAG AGATGCGCTT CCTGTCACGC ATCTCTTTCA GTCACTTCCT TTTTTggaaaC -3', siFlk1-C bottom: 5'- tcgaGtttcc AAAAAAGGAA GTGACTGAAA GAGATGCGTG ACAGGAAGCG CATCTCTTTC AGTCACTTCCG -3'.
Flow Cytometry Analysis
To dissociate the EBs, floating EBs were incubated with 0.25% trypsin and 1 mM of EDTA (Invitrogen) at 37°C for 1–3 minutes with occasional agitation. As soon as the solution became cloudy, FCS-containing medium was added to neutralize the trypsin. After centrifugation, single cells were resuspended in phosphate-buffered saline containing 2% FCS and 2.5 µg/ml of propidium iodide (PI) or 1 µg/ml of 7-amino actinomycin D (7AAD) to stain the dead cells, and then analyzed with a FACS CaliburTM or FACS Vantage SETM (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com), as described previously [17]. To detect Flk1-positive cells, cells were dissociated from EBs as described above and incubated with anti-Flk1 antibody (BD Biosciences) conjugated with phycoerythrin (PE).
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RESULTS
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Site-Directed Integration of siRNA Expression Cassette at Hprt Locus
Constitutively active housekeeping gene loci, such as the Hprt locus and ROSA26 locus, are often used to introduce transgenesis in murine ESCs by homologous recombination [24, 25]. To use the Hprt locus, we deleted the first exon of the Hprt gene, using an Hprt-deleting vector by homologous recombination, as described previously [15]. We also inserted the EGFP reporter gene into the ROSA26 locus to ensure reporter expression over a long term [26]. The resultant Hprt–EGFP+ ESC line (termed hgh2; Fig. 1A), was then transfected with the Hprt targeting vector (pHprt-siGFP; Fig. 1B), which contained the human H1-promoter–driven siRNA expression cassette for the EGFP gene. Because the Hprt targeting vector carries the first and second exons of the Hprt gene, only homologous recombined clones were rescued and became Hprt+ (Fig. 1C). Among the HAT-resistant colonies that appeared on day 10, nearly all exhibited much less fluorescence as compared with the hgh2 cells, as assessed under a fluorescence microscope (Fig. 2A). Five randomly selected clones were found to contain only one copy of the human H1 promoter sequence, as determined by quantitative PCR of genomic DNA (data not shown), suggesting that the siRNA cassette was solely integrated into the Hprt locus and not integrated randomly into the genome. As shown in Figure 2B, these five clones expressed approximately 30-fold lower GFP fluorescence than the hgh2 cells, suggesting that suppression by the siRNA cassette integrated at the Hprt locus was highly reproducible and homogeneous.
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Figure 1. Introduction of siRNA expression cassette at the Hprt locus. (A): The Hprt–EGFP+ cell line, hgh2, is depicted. It constitutively expresses EGFP driven by the CAG promoter at the ROSA26 locus. In this cell line, the genomic region containing the first exon of the Hprt gene was replaced with a neomycin-resistant gene cassette (MC1neo). (B): Schematic design of the Hprt targeting vector containing the first and second exons of the Hprt gene is shown. The red box indicates the siRNA expression cassette, containing the H1 promoter and the short hairpin small interfering sequence. The short hairpin sequence for EGFP, indicated in the lower part, is derived from pGtoR. (C): The siRNA-expressing ESC derived from hgh2 is depicted. By homologous recombination with the Hprt targeting vector, the Hprt locus is reconstituted to confer Hprt positivity and the siRNA cassette for GFP is integrated at the Hprt locus. Abbreviations: EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GFP, green fluorescent protein; siRNA, small interfering RNA.
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Figure 2. Long-term suppression of EGFP in five in dependent clones. (A): Fluorescence of siGFP-expressing ESCs. The Hprt–EGFP+ ESC line, hgh2, was transfected with the Hprt targeting vector containing siRNA cassette for EGFP. Nearly all Hprt+ clones exhibited reduced expression of EGFP. One of the siGFP-expressing clones (#1), hgh2, and its parental strain, ht7, are shown. (B): Relative intensity of GFP fluorescence of siGFP-expressing ESCs (#1–#5) before (passage 1) and after (passage 10) long-term culture. Mean fluorescence intensity of hgh2 obtained from flow cytometry analysis is set as 100%. Abbreviations: EGFP, enhanced green fluorescent protein; ESC, embryonic stem cell; GFP, green fluorescent protein; PI, propidium iodide; siGFP, small interfering RNA–expressing cassette for GFP; siRNA, small interfering RNA.
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To test the stability of the suppression effect, we cultured ESCs expressing siRNA for 10 passages without HAT selection and analyzed GFP fluorescence by flow cytometry. As indicated in Figure 2B
, a suppressing effect was observed after approximately 1 month without any significant decrease, indicating that the expression of the siRNA cassette from the Hprt locus was highly stable and not subject to an epigenetic effect.
Gene Suppression in Differentiated Cells Derived from ESCs
The siGFP-expressing ESCs underwent multiple rounds of transfection; therefore, it was considered important to determine if they retained the ability to differentiate into multiple lineages of cells. The siGFP-expressing ESCs formed EBs as efficiently as the control ESC clones. Furthermore, the efficiency for production of EBs that exhibited spontaneous beating was nearly comparable to that of the original cell line, ht7 (Fig. 3A), indicating that the cardiomyogenic potential was retained. We also observed multiple cell types, such as smooth muscle, cardiac muscle (Fig. 3B), endothelial-like, and neuron-like cells (data not shown) that were derived from the siGFP-expressing ESCs. Thus, the siGFP-expressing ESCs retained a differentiation potential comparable to that of the original ESCs.
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Figure 3. Differentiation of EBs derived from stably transfected cells. (A): Frequency of spontaneous beating EBs derived from siGFP-expressing ESCs. EBs were formed by the hanging drop method. On day 5 of differentiation, EBs were transferred onto 24-well gelatin coated plates and the number of spontaneous beating EBs was counted. Three independent differentiation experiments were performed for each clone. (B): Differentiation of siGFP-expressing cells into sarcomeric myosin heavy chain–positive or SM actin–positive cells. The siGFP-expressing EBs were dissociated and replated onto a gelatin-coated 35-mm dish. Immunocytochemical analysis was performed using anti-sarcomeric myosin heavy chain antibody (MF20; Developmental Studies Hybridoma Bank, Iowa City, IA, http://www.uiowa.edu/~dshbwww) and anti-SM- -actin antibody (1A4; Sigma, St. Louis, http://www.sigmaaldrich.com) after incubation with the secondary antibody conjugated with Alexa546 (Molecular Probes, Inc., Eugene, OR, http://probes.invitrogen.com). Abbreviations: EB, embryoid body; ESC, embryonic stem cell; GFP, green fluorescent protein; siGFP, small interfering RNA–expressing cassette for GFP; SM, smooth muscle.
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Those cells differentiated from EBs that were derived from siGFP-expressing cells exhibited a much weaker fluorescence than cells derived from the parental ESCs (hgh2), as assessed with a fluorescence microscope (Fig. 4A). To quantify the suppressive effect of RNAi in the differentiated cells, EBs derived from five independent clones were dissociated and analyzed by flow cytometry. On average, the siGFP-expressing ESC-derived cells exhibited an approximately 10-fold lower level of fluorescence than hgh2 cells (Fig. 4B). Furthermore, there were no significant differences in the levels of suppression among the individual clones, whereas each exhibited a similar GFP fluorescence histogram profile (data not shown). The histogram pattern of each clone also suggested that RNAi occurred uniformly in each differentiated cell, though not in specific patterns in individual cell types. These results suggested that the suppression of GFP by the siRNA cassette was equally effective in each stage of differentiation of ESCs.
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Figure 4. Gene suppression of GFP in differentiated cells. (A): Fluorescence of EBs derived from siGFP-expressing ESCs. EBs were formed as described in Results. GFP fluorescence was observed under a fluorescence microscope. (B): Relative intensity of GFP fluorescence of siGFP-expressing ESC-derived cells. Mean fluorescence of hgh2 obtained from flow cytometry analysis is set as 100%. Abbreviations: EB, embryoid body; ESC, embryonic stem cell; GFP, green fluorescent protein; siGFP, smal interfering RNA–expressing cassette for GFP.
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Suppression of Endogenous Genes by siRNA Cassette Integrated at the Hprt Locus
To test if the siRNA expression system also functions to suppress endogenous genes in ESCs, we designed three different siRNA cassettes for the Flk1 (Kdr) gene (siFlk1-A, -B, and -C) and integrated them into the Hprt locus of Hprt-ESCs (hdh31) using the Hprt targeting vector. The Flk1 gene encodes vascular endothelial growth factor receptor 2 and is expressed in nascent mesoderm, hemangioblast, and endothelial cells derived from ESCs [27]. Typically, more than 30% of the ESCs derived from wild-type ESCs (ht7) became Flk1-positive on day 5 (Fig. 5A). Whereas one of the siRNA cassettes (siFlk1-A) did not seem to be effective in suppression of the Flk1 gene, the two others (siFlk1-B and -C) efficiently suppressed its expression. As shown in Figure 5B, only 2%–4% of the cells expressing siFlk1-B or -C were Flk1-positive. The Flk1-positive cells derived from siRNA-expressing ESCs exhibited weaker fluorescence as compared with cells derived from wild-type ESCs (approximately 50% reduction for siFlk-B or -C). These results suggest that siRNA from the Hprt locus can suppress endogenous genes efficiently and stably in ESCs.
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Figure 5. Gene suppression of Flk1 in differentiated cells. (A): Flow cytometry analysis of siFlk1-expressing ESC-derived EBs. The ht7-derived Hprt– ESC line, hdh31, was transfected with the Hprt targeting vectors with three different designs of siRNA cassette for Flk1 (siFlk1-A, -B, and -C). The Hprt+ clones were selected, expanded, and differentiated by the method of EB formation. EBs were dissociated by trypsin-EDTA on day 5, stained with anti-Flk1 antibody conjugated with PE. Dead cells were stained with 7AAD. Flow cytometry analyses of ht7– and siFlk1-B–expressing cells are shown in the left and right frames, respectively. (B): Frequency of Flk1-positive cells in ht7- and siFlk1-expressing EBs (left). Two independent clones for each siFlk1 cassette (siFlk1-A, -B, and -C) were examined, and similar results were obtained (data not shown). Relative intensity of PE fluorescence in the Flk1-positive cell fraction of siFlk1-expressing EBs (right). Mean fluorescence of Flk1-PE–positive cell fraction (R2 in Figure 5A) of ht7 is set as 100%. Abbreviations: 7AAD, 7-amino-actinomycin D; EB, embryoid body; ESC, embryonic stem cell; PE, phycoerythrin; siRNA, small interfering RNA.
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DISCUSSION
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ESCs provide a useful model system for studying cell differentiation in vitro; however, functional analysis has been hampered by difficulties in modulating the expression of specific genes. Recent advances in RNAi technology have made it possible to suppress specific targets by introduction of an siRNA vector into ESCs, though in past studies, the siRNA expression cassette was randomly integrated into the chromosomes. Because random integration of a transgene generally results in different expression levels, due to the positional effect, it is necessary to examine multiple transgenic clones to evaluate the suppressing effect. In the approach used in the present study, the siRNA expression cassette was integrated into the Hprt locus in a site-specific manner, and we confirmed our speculation that the reproducibility and uniformity of the suppressive effect occurred in individual ESC clones before and after differentiation. Therefore, this method could indeed avoid the problems of variable expression of the gene cassette and multiple experimental steps for cell selection. Although the frequency of homologous recombination events is generally much lower than that of random integration, successful targeting can be easily accomplished by growing cells in HAT medium. Because most HAT-resistant clones contain the correct construct, only a few clones are necessary to examine the effect of an siRNA. Therefore, the present method should provide a great advantage for screening potentially important genes with an in vitro differentiation system, though possible disadvantages include gene silencing of the locus.
Recent studies have demonstrated that hypomorphic changes in gene expression result in phenotypes that are sometimes associated with those of human diseases [28]. Unlike a gene knockout method, siRNA-based technology can easily alter the expression of specific genes to obtain a hypomorphic type [29], though it may be laborious to select moderately effective siRNA sequences by random integration of the siRNA cassette, which is subject to a positional effect. Because the present system provides a uniform suppressive effect, it is possible to choose an appropriately effective siRNA before generating ESC-derived mice. Thus, the knockdown technique described here is potentially quite useful for generating valuable animal models in which the level of gene expression is finely modulated, which may lead to new insights into the functions of genes related to human diseases.
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ACKNOWLEDGMENTS
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We express our thanks to H. Niwa for providing the ht7 ESCs, S.K. Bronson for plasmid pHprt, S. Duncan for plasmid pMP8NEBlacZ, M. Okabe for plasmid pGtoR, J. Miyazaki for plasmid pCX-EGFP, and P. Soriano for plasmid pROSA26-1. This work was supported in part by the Promotion of Fundamental Studies in Health Science of the Organization for Pharmaceutical Safety and Research (OPSR) of Japan, by Research Grants on Cardiovascular Diseases from the Ministry of Health, Labor, and Welfare, Japan, and by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Sciences (JSPS).
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Received November 28, 2004;
accepted for publication June 2, 2005.
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Abstract
Introduction
