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Stem Cells 2004;22:93-99 www.StemCells.com
© 2004 AlphaMed Press

Stable Suppression of Gene Expression in Murine Embryonic Stem Cells by RNAi Directed from DNA Vector-Based Short Hairpin RNA

Fu-Chou Tang, Guo-Liang Meng, Hong-Bo Yang, Cheng-Jian Li, Yan Shi, Ming-Xiao Ding, Ke-Gang Shang, Bo Zhang, You-Fang Xue

College of Life Sciences, Peking University, Beijing, P. R. China

Key Words. ES cell • RNAi • U6 promoter • Short hairpin RNA • Green fluorescent protein

You-Fang Xue, M.D., Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, 100871, P. R. China. Telephone: 86-10-6275-1858; Fax: 86-10-6288-5710; e-mail: zhouym{at}pku.edu.cn


   ABSTRACT
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Murine embryonic stem (ES) cells are an ideal system for the research of directed differentiation in vitro. Long double-stranded RNA, which can induce RNA interference (RNAi) effectively in many organisms, has been shown to suppress target gene expression efficiently and specifically in undifferentiated ES cells. However, it cannot be used in differentiated ES cells due to unspecific inhibition of gene expression resulting from the activation of interferon pathway following differentiation. Using green fluorescent protein (GFP) as a reporter system, we show here that a short hairpin RNA (shRNA) expression vector driven by the murine U6 small nuclear RNA promoter can specifically induce potent gene knockdown effect (i.e., inhibit GFP expression specifically) when transfected transiently into ES cells. Furthermore, when the expression vector is stably integrated into the genome of the cell, it can still show specific RNAi effect, which can be maintained at least for 10 days. These transfected ES cells showed no obvious differences in the morphology or growth rate in culture compared with untransfected cells, suggesting that the activation of shRNA-directed RNAi did not affect the properties of ES cells and that the RNAi effect in ES cells is specific and persistent. Our results prove the feasibility of the U6 promoter-driven shRNA expression technique to be used to study the function of genes expressed in ES cells. These ES cells, after integration of the U6-based RNAi vector into their genome, could be used to generate gene knockdown mice.


   INTRODUCTION
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Murine embryonic stem (ES) cells are an ideal model for the research of mammalian cell differentiation in vitro [1]. Recently, these cells have been widely used in directed differentiation into several kinds of cells, including neurons, pancreatic cells, and hematopoietic cells. [2–5]. The rate of differentiation can be stimulated by inhibiting or inducing the expression of certain genes [6]. Currently, there are two ways to achieve targeted gene suppression in ES cells: homologous recombination (HR)-based gene knockout and double-stranded RNA (dsRNA)-dependent gene knockdown [7], which is commonly called RNA interference (RNAi). The HR-based gene knockout method has been well developed and has proved to be a powerful tool to understand the function of genes in ES cells, especially in whole mice. Although it can eliminate target gene expression completely, the method itself is usually time consuming and laborious. In addition, usually only one of the two alleles of the gene can be deleted in ES cells at one time. To generate gene knockout ES cells, another round of HR is required to delete the second allele. On the other hand, since the knockout method generally results in a null mutation, it is difficult to study the dosage effect of a gene.

RNAi, which was discovered in recent years, is a method of gene silencing involving dsRNA [8]. dsRNA can specifically and efficiently inhibit gene expression by degrading corresponding mRNA. Simple and easy to control, RNAi has been used successfully to knockdown gene expression in Caenorhabditis elegans and Drosophila melanogaster on a large scale [9]. Unfortunately, due to certain limitations, the RNAi technique so far is not well established in higher organisms such as mammals. Recently, it was shown that long dsRNA could be used to knockdown gene expression in undifferentiated murine ES cells [10–12]. However, as soon as the ES cells differentiate, the interferon pathway would be activated and dsRNA longer than 30-bp would cause the global shutdown of protein synthesis, leading to unspecific gene suppression [13]. This unspecific effect can be avoided by using synthesized small interfering RNA (siRNA), which is less than 30-bp. However, commercial RNA oligonucleotide synthesis is very expensive and the silencing effect is transient, i.e., the knockdown effect can be maintained only for about 1 week [14].

Very recently, a hairpin siRNA expression system driven by RNA polymerase III-dependent U6 small nuclear RNA promoter was shown to be able to suppress target gene expression in mouse p19 carcinoma cells as well as in human cells [15–19]. This certainly provides an inexpensive and easy way to bypass the unspecific response from long dsRNA and raises the possibility for long-term target gene silencing by RNAi in whole organisms such as mice. For this purpose, the silencing effect and efficiency of U6-driven short hairpin RNA (shRNA) in ES cells and, more importantly, the long-term persistence of this effect have to be tested. Here we show that the U6-based RNAi system works very well in murine ES cells. Furthermore, when the expression vectors are integrated into the genome of the cell, they still show potent gene knockdown effect that can be maintained for at least 10 days. Our results proved the feasibility of vector-based RNAi directed by shRNA to induce potent and heritable RNAi effect in murine ES cells. This provides a simple alternative to the traditional knockout method to explore the biological function, as well as the dosage effect, of differentiation-related genes in the process of directed differentiation of ES cells. These results also provide the possibility to use shRNA-directed gene knockdown ES cells to study gene function in vivo simply by generating gene knockdown mice through these ES cells.


   MATERIALS AND METHODS
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Culture of ES Cells
The ES cell line MESPU13 from the 129/ter mouse strain was established and characterized in our laboratory [20]. Its properties and culture conditions have been described previously [20]. Briefly, the ES cells were grown on feeder cells (1 ~ 5 x 105 per 60-mm culture dish) derived from mitomycin C (100 µg/ml, Sigma; http://www.sigmaaldrich.com; St. Louis, MO)-treated murine primary embryonic fibroblasts and were maintained and propagated in plastic tissue culture dishes (coated with 0.1% gelatin) in Dulbecco’s modified Eagle’s medium (high glucose, 4.5 g glucose/l with 2 mM L-glutamine without pyruvate [GIBCO; Bar Harbor, ME; http://www.lifetech.com]) supplemented with 20% heat-inactivated fetal calf serum (selected batch from Tianjin, China) at 37°C in humidified air/5% CO2. Leukemia inhibitory factor (Chemicon; Temecula, CA; http://www.chemicon.com) at a final concentration of 1,000 U/ml and 0.1 mM ß-mercaptoethanol were added to standard medium to prevent differentiation of ES cells. The cells were regularly split every 2 days and kept in culture no longer than 10 passages.

Construction of Expression Vectors
The plasmid pCG-UH (Fig. 1Go), which contains a green fluorescent protein (GFP) expression cassette (under the control of cytomegalovirus promoter) and an shRNA (against GFP) producing cassette (under the control of U6 promoter), was constructed by inserting the 0.8-kb Hind III-Pvu II fragment from pU6-GFP-HP28 (an shRNA expression vector against GFP under the control of U6 promoter, a kind gift from Dr. D.L. Turner, University of Michigan) [19] into the Afl II site of pEGFP-N2 (Clontech; Palo Alto, CA; http://www.bdbiosciences.com/clontech) [19]. The control plasmid pCG-U (Fig. 1Go), which contains the GFP expression cassette and an empty U6 promoter, was constructed by inserting the 0.8 kb Sal I-EcoR I fragment of pmU6pro (containing an empty U6 promoter: the same as pU6-GFP-HP28 except that there is no sequence corresponding to shRNA following U6 promoter, used as a control vector for pU6-GFP-HP28, a kind gift from Dr. D.L. Turner) [19] into the Afl II site of pEGFP-N2 [19]. The target site for the silencing of GFP is: 5'-GAAGCAGCACGACTTCTTC-3'. For stable integration into the genome of ES cells, the plasmids were linearized by ApaL I, purified by agarose gel electrophoresis, and recovered by adsorption onto glass beads. All of the plasmids contain G418-resistant gene as a selection marker for stable integration into ES cells.



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  		Figure 1. Map of constructs used in this work. pU6-GFP-HP28 contains the shRNA (i.e., shGFP) expression cassette driven by the U6 promoter. It was used for the cloning of the combined GFP expression and shRNA-producing vector pCG-UH. pmU6pro is a control plasmid for pU6-GFP-HP28. It is basically the same as pU6-GFP-HP28 except that there is no sequence-encoding shRNA following the U6 promoter. This plasmid was used to construct our control vector pCG-U. pCG-UH contains both the full-length GFP expression cassette driven by the cytomegalovirus (CMV) promoter (from pEGFP-N2) and the shRNA expression cassette against GFP (from pU6-GFP-HP28), and was used as a combined reporter and interference plasmid to test the RNAi effect in ES cells in both transient transfection and stable integration experiments. pCG-U was used as a control vector for pCG-UH since it lacks the shRNA-encoding sequence compared with pCG-UH. pEGFP-N2 was used to provide full-length GFP expression cassette for the construction of pCG-UH and pCG-U.

 
Polymerase Chain Reaction of Genomic DNA
The ES cells were harvested from a 24-well culture plate by trypsin-EDTA digestion and lysed at 50°C for 8–12 hours in lysis buffer (50 mM KCl, 10 mM Tris-Cl [pH 8.3], 2 mM MgCl2, 0.45% NP-40, 0.45% Tween-20, 100 µg/ml proteinase K). Proteinase K was inactivated at 97°C for 30 minutes, and the lysis product was used directly as a polymerase chain reaction (PCR) template. The PCR primer sequences for GFP are as follows: 5'-CTCGTGACCACC CTGACCTA-3' (sense); 5'-GCTCAGGTAGTGGTTGTC GG-3' (antisense). These sequences are located outside the silencing target region of GFP. The expected size of GFP fragment from the vectors is 429 bp.

Transient Transfection of ES Cells by Lipofectin
The transfection was performed in 24-well culture plates. Two micrograms of plasmid DNA and 10 µl lipofectin (Invitrogen; Carlsbad, CA; http://www.invitrogen.com) were used for each well. The ES cells were transferred into fresh medium without lipofectin or plasmids and continued in culture for a further 72 hours. The cells were trypsinized into single cell suspension and the GFP activity was assessed by measuring cell fluorescence through flow cytometry (fluorescence-activated cell sorting [FACS] analysis). In every transfection, 30,000 events were saved for each FACS run to calculate the number and percentage of GFP-positive cells. The same set of experiments was repeated three times independently.

Production of Stably Transfected ES Cell Clones
Stable integration of DNA vectors into ES cells was achieved by electroporation according to standard protocol (250 V, 500 µF) [21]. Twenty-five micrograms linearized plasmid DNA was used for 5.6 x 106 ES cells. G418 (Geneticin, Invitrogen) at 250 µg/ml was used to select integrated clones. During 10 days of selection, the green fluorescence was monitored under blue excitation light through a fluorescence microscope and then the resistant clones were picked up randomly for further experiments and amplified following standard ES cell culture conditions.


   RESULTS
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
We chose GFP as our reporter system to test the effect of a GFP-RNA-directed shRNA expression vector. We wanted to observe both short-term and long-term effects by transient transfection and stable integration of the shRNA expression plasmid, respectively. To rule out the influence of copy numbers or position effects in stable integration experiments, we integrated our reporter plasmid (pEGFP-N2) and effector plasmid (pU6-GFP-HP28) into one construct called pCG-UH by inserting the shRNA expression cassette (including shRNA-encoding sequence against GFP and U6 promoter) into the plasmid pEGFP-N2 (Fig. 1Go). As a control, we constructed another vector (pCG-U, by inserting pmU6pro into pEGFP-N2) that is identical to pCG-UH except that there is no sequence corresponding to shRNA of GFP after the U6 promoter (Fig. 1Go).

We first determined if the U6 promoter-driven shRNA could induce specific RNAi effect in murine ES cells by transient transfection. pCG-UH and pCG-U were separately introduced into ES cells by lipofectin transfection. Seventy-two hours after transfection, the cells were harvested and the ratio of GFP-expressing cells to whole cell population was calculated by flow cytometry. Although the transfection efficiency is quite low (only about 15%), there was still an obvious difference in the percentage of GFP-positive cells between the transfection with pCG-UH and pEGFP-N2 (Table 1Go, 8.3% ± 1.6% versus 14.5% ± 2.5%, respectively). Transfection of shRNA expression cassette containing construct pCG-UH obviously reduced the amount of GFP-positive cells, suggesting that shRNA against GFP was expressed in ES cells and its expression led to the inhibition of GFP expression (Table 1Go). When control vector pCG-U was used for transfection instead of pCG-UH, the percentage of GFP-positive cells did not show such a difference (Table 1Go, 13.7% ± 2.2% versus 14.5% ± 2.5%, respectively). This means that the sequence-encoding shRNA led to the reduction of GFP expression. Cotransfection of pU6-GFP-HP28 and pEGFP-N2 into ES cells gave similar RNAi results compared with the transfection with the integrated plasmid pCG-UH (data not shown). These results show that U6 promoter-driven shRNA, when expressed transiently in murine ES cells, could induce a specific RNAi effect.


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  		Table 1. The ratio of green fluorescent cells in transient transfected ES cells*
 
Next, we determined whether the shRNA expression vector could induce similar RNAi effect when integrated into the genome of ES cells. We electroporated pEGFP-N2, pCG-U, and pCG-UH into ES cells separately. G418 was used as a selection marker for stable integration of the plasmids. After 10 days of selection, 300 resistant clones were chosen randomly from each experimental group and counted for GFP-positive clones using the fluorescence microscope. The same set of experiments was repeated three times independently. In the ES cells that were electroporated by pEGFP-N2, approximately 71.3% ± 20.3% clones were GFP positive (defined as >=10% of the clone area showing green fluorescence) (Fig. 2A–2DGo; Table 2Go), whereas only 9.2% ± 2.3% clones showed positive green fluorescence when pCG-UH was used in electroporation (Fig. 2I–2LGo; Table 2Go). Among the positive clones, the overall green fluorescent signal was much weaker than in the former group. As a control, when pCG-U was used for electroporation, there were 78.3% ± 13.0% GFP-positive clones (Fig. 2E–2HGo; Table 2Go). From these results we concluded that the U6-driven shRNA expression system is also effective to mediate RNAi after stable incorporation into the genome of ES cell, and more importantly, the gene suppression effect is still specific.



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  		Figure 2. GFP expression in ES cells stably integrated with the plasmid pEGFP-N2, pCG-U, or pCG-UH after a 10-day selection by G418 and subsequent 10-day amplification. A-L) GFP expression in G418-resistant ES clones after 10-day selection. A-D) the ES clones stably integrated with the plasmid pEGFP-N2; E-H) the ES clones stably integrated with the plasmid pCG-U; I-L) the ES clones stably integrated with the plasmid pCG-UH. C, D, G, H, K, L) green fluorescent image showing GFP expression; A, B, E, F, I, J) bright field microscopic image corresponding to C, D, G, H, K, L, respectively. Original magnification 100x. M-X) GFP expression in ES clones after a 10-day amplification. All colonies in one picture are derived from a single ES clone and have the same genetic background. M-P) the ES clones stably integrated with the plasmid pEGFP-N2; Q-T) the ES clones stably integrated with the plasmid pCG-U; U-X) the ES clones stably integrated with the plasmid pCG-UH. O, P, S, T, W, X) green fluorescent image showing GFP expression; M, N, Q, R, U, V) bright field microscopic image corresponding to O, P, S, T, W, X, respectively. Original magnification 100x.

 

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  		Table 2. The ratio of green fluorescent clones in stably transfected ES cell clones*
 
We further inquired if the gene knockdown effect by RNAi is heritable, or in other words, if the gene knockdown effect could be maintained stably during passages. For this purpose, 24 clones were picked up randomly from each electroporation group and amplified in culture for up to 10 days (equal to five passages). In the group with pEGFP-N2, 16 of 24 clones (67%) emitted partial or full green fluorescence under the fluorescence microscope throughout the amplification (Fig. 2M–2PGo). However, in the group of electroporation with pCG-UH, only three clones (13%) showed GFP positivity at the beginning of amplification. At the end of five passages, the same positive clones emitted green fluorescence under the same bright blue activation; most of the clones were GFP negative (Fig. 2U–2XGo). As a control, in the group of electroporation with pCG-U (Fig. 2Q–2TGo), there were 18 clones (75%) that exhibited green fluorescence under the same amplification conditions. This percentage of GFP-positive clones was comparable to the pEGFP-N2 electroporation group. During these five passages, there were no visible changes in the fluorescent state in all groups, suggesting that the specific GFP gene suppression effect can be maintained stably under our experimental conditions (data not shown). Compared with untransfected ES cells, no obvious visible differences in morphology or growth rate were observed in all these ES cell clones (Fig. 2Go). This suggests that the activation of shRNA-directed RNAi effect did not affect the properties of ES cells, and the gene knockdown effect is both specific and persistent.

To rule out the possibility that failure in genomic integration or loss of the transgene led to the loss of green fluorescence, we checked by PCR the genomic DNA of clones from the above experiments (Fig. 3Go). The results showed that in nearly all of the clones the GFP transgene still exists (pEGFP-N2: 23 of 24 showed positive; pCG-U: 22 of 24 showed positive; pCG-UH: 24 of 24 showed positive). This provides sound evidence that the lack of green fluorescence was not due to failure in plasmid integration or the selective loss of the vector pCG-UH during passages.



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  		Figure 3. PCR analysis of ES cell clones after amplification. Results of part of the transgenic ES clones were shown. Md) DNA marker DL2000, the size of the 6 bands is: 2.0 kb, 1.0 kb, 0.75 kb, 0.5 kb, 0.25 kb, 0.1 kb, respectively; Mi) DNA marker MII, the size of the 6 bands is: 1.2 kb, 0.9 kb, 0.7 kb, 0.5 kb, 0.3 kb, 0.1 kb, respectively; N) negative control without DNA template added; 1–8) genomic DNA templates from ES cell clones integrated with pEGFP-N2; 9–16) genomic DNA templates from ES cell clones integrated with pCG-U; 17–26) genomic DNA templates from ES cell clones integrated with pCG-UH. The expected size of the band for GFP in all three plasmids is 429 bp (arrow).

 

   DISCUSSION
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
RNAi induced by U6 promoter-directed shRNA is not equally efficient in all mammalian cell lines tested so far [22]. Usually, it is necessary to use a transfected reporter gene to determine the efficiency of shRNA in inhibiting target gene expression in a given cell line [13–19]. However, in many cells the transfection efficiency is too low to assess gene knockdown effect by cotransfection of the reporter gene and shRNA expression vector. The vector (i.e., pCG-UH) we constructed here, which can express functional full-length GFP protein and transcribe shRNA corresponding to part of the GFP sequence, somehow overcomes the problem. Even when the transfection efficiency is low, it is still easy to detect whether specific gene suppression has happened.

RNAi is a phenomenon of gene silencing directed by dsRNA [8]. It can specifically inhibit gene expression by degrading mRNA efficiently and has been widely used to knockdown gene expression in genomes such as Caenorhabditis elegans and Drosophila melanogaster [9]. For mammalian cells, dsRNA-directed RNAi was detected only in murine undifferentiated ES or embryonic carcinoma cells [10–12]. Our previous work also proved the existence of RNAi effect for both exogenous reporter gene GFP and endogenous gene Oct4 in undifferentiated ES cells [12]. However, in other kinds of mammalian cells, because of the existence of the interferon pathway, long dsRNA could induce the cells to shut down global protein translation and activate apoptosis, leading to unspecific gene silencing. Therefore, dsRNA longer than 30-bp cannot be used to induce specific gene knockdown effect in these cells [13]. Elbashir et al. reported that in vitro synthesized, 21-23-nt siRNA could induce a potent RNAi effect as effective as long dsRNA without showing the unspecific effect, so that the interferon pathway could be bypassed [14]. It was shown that during the RNAi process, long dsRNA was first degraded into 21-23-nt siRNA and then recruited into RNA-induced silencing complex to degrade corresponding mRNA. However, the synthesis of siRNA is expensive and the effect is transient because the knockdown effect can only be maintained for about 1 week [14].

Recently, it has been shown that a U6-promoter-driven shRNA could induce potent gene knockdown effect in several kinds of mammalian cells [13–19]. It is not known, however, if this also works well when the plasmid is integrated into the genome of the cell since only transient transfection of the shRNA plasmid has been tested so far. This is especially interesting for ES cells since it could provide a simple and easy way to generate gene functional knockout mice if persistence of the RNAi gene knockdown effect in ES cells was achieved [23]. For this purpose, we tested the RNAi effect of a U6-promoter-driven shRNA in murine ES cells using GFP as our reporter gene. First, we showed that when transiently transfected into ES cells, this shRNA expression vector could inhibit GFP expression specifically and efficiently, yet the effect of shRNA, when the vector is integrated into ES cell genome, is obviously much more important. Our results showed that after stable integration, U6 promoter-driven shRNA could still specifically inhibit GFP expression with comparable efficiency to that of transient transfection. Furthermore, the integration of the vector and the persistent expression of the shRNA did not affect the morphology, proliferation, or other characteristics of ES cells. Our data proved the feasibility of vector-based RNAi directed by shRNA to induce potent and heritable RNAi in murine ES cells. It also provides the possibility of using RNAi transgenic ES cells by U6-promoter-directed shRNA to produce gene knockdown mice and to study corresponding gene function in vivo.


   ACKNOWLEDGMENT
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The authors would like to thank Dr. D.L. Turner for kindly providing the plasmids pU6-GFP-HP28 and pmU6pro, and Heather Owen for critical reading and editing of English. This work was supported in part by a grant for "Study of RNA Interference in Murine Embryonic Stem Cells (No. 30170456)" from the National Natural Science Foundation of China.


   REFERENCES
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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  9. Bosher JM, Labouesse M. RNA interference: genetic wand and genetic watchdog. Nat Cell Biol 2000;2:E31–E36.[CrossRef][Medline]

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  11. Paddison PJ, Caudy AA, Hannon GJ. Stable suppression of gene expression by RNAi in mammalian cells. Proc Natl Acad Sci USA 2002;99:1443–1448.[Abstract/Free Full Text]

  12. Meng G, Tang F, Zhang J et al. RNA interference in three murine ES cell lines from different mouse strains. Acta Biochimic et Biophysic Sinica 2003;35:238–246.

  13. Paddison PJ, Caudy AA, Bernstein E et al. Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells. Genes Dev 2002;16:948–958.[Abstract/Free Full Text]

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Received January 31, 2003; accepted for publication September 29, 2003.



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