Kinetics of drug selection systems in mouse embryonic stem cells
© Nakatake et al.; licensee BioMed Central Ltd. 2013
Received: 18 March 2013
Accepted: 5 August 2013
Published: 7 August 2013
Stable expression of transgenes is an important technique to analyze gene function. Various drug resistance genes, such as neo, pac, hph, zeo, bsd, and hisD, have been equally used as selection markers to isolate a transfectant without considering their dose-dependent characters.
We quantitatively measured the variation of transgene expression levels in mouse embryonic stem (mES) cells, using a series of bi-cistronic expression vectors that contain Egfp expression cassette linked to each drug resistant gene via IRES with titration of the selective drugs, and found that the transgene expression levels achieved in each system with this vector design are in order, in which pac and zeo show sharp selection of transfectants with homogenously high expression levels. We also showed the importance of the choice of the drug selection system in gene-trap or gene targeting according to this order.
The results of the present study clearly demonstrated that an appropriate choice of the drug resistance gene(s) is critical for a proper design of the experimental strategy.
The introduction of exogenous transgene cassettes into culture cells to direct their expressions is an important strategy in molecular biology to analyze the functions of the genes. However, a simple introduction of the DNA fragment into cells by either electroporation or lipofection results in its stable integration into the genome of the host cells only at a low frequency. Therefore, it is always required to select the cells carrying the integrated copies of the transgenes by using dominant selection markers. The combinations of the antibiotics that kill the mammalian cells and the genes that establish the resistance against them have been preferentially applied for this purpose: such as neomycin phosphotransferase II from transposon Tn5 (designated as neo in this paper) against the neomycin derivative G418, puromycin N-acetyltransferase from Streptomyces alboniger (pac) against puromycin, hygromycin B phosphotransferase from Escherichia coli (hph) aginst hygromycin B, Streptoalloteichus hindustanus ble (Sh ble: designated as zeo in this paper) against the bleomycin derivative zeocin, blasticidin S deaminase from Aspergillus terreus (bsd) against blasticidin S, and histidinol dehydrogenase from Salmonella typhimurium (hisD) against histidinol [1–6]. These drugs and the resistance genes have equally been regarded as dominant selection markers that reflect the introduction of the transgenes into mammalian cells. Transfection of drug resistance genes together with transgenes, each in separate expression cassette, to obtain stable transfectants has been a commonly used method. However, in this strategy, the drug resistance does not always appropriately reflect the expression level of the transgene because generally the stable expression levels of exogenous expression cassettes are highly sensitive to thier sites of integration, as a result of the local chromatin environment when the transgenes are randomly integrated into the host genome , which affect the expression levels of the drug resistance gene cassette and the transgene cassette separately.
The bi-cistronic expression of the transgene and the drug resistance gene using an internal ribosome entry site (IRES) is able to confer a tight correlation between the transgene expression and the drug resistance because the IRES-mediated cap-independent translation ensures parallel expressions of the transgene and the drug resistance gene . This vector design is particularly important to drive transgene expressions in mouse embryonic stem (mES) cells since the silencing effect to the stably integrated transgene cassette is problematic in these cells . In this vector design, the expression levels of the transgenes depend on the threshold expression levels of the drug resistance genes that confer the survival of the transfectants in the presence of the drugs. The expression levels of some transgenes can also possess unique thresholds based on their effect on cell viability. The combination of the effect of the transgene with the range of selection generated by the antibiotic resistance marker can produce a narrow expression range that can mimic the physiological function range of expression. Therefore, a proper choice of drug resistance genes is important to achieve the optimal range of transgene expression levels.
Here we demonstrated the parallel comparison of the kinetics among each drug selection system determined by the expression levels from the IRES-based expression vectors. We found obvious differences in their kinetics and the impact on various experimental situations.
Kinetics of the drug-selection systems in the IRES-based expression vectors
Generation of new fusion genes of fluorescent markers and drug-resistant genes
Application of the drug-resistant systems to gene targeting
The expression levels of the genes targeted by the promoter-less vectors in ES cells
pac, hph, bsd
Toyooka et al. , 2008 []
Masui et al. , 2008 []
pac, neo, zeo
Nichols et al. , 1998 []
Toyooka et al. , 2008 []
Masui et al. , 2007 []
Taniguchi et al. , 2009 []
Takashima et al. , 2002 []
Vitaterna et al. , 1999 []
Tateishi et al. , 2003 []
Vitaterna et al. , 1999 []
Martello et al, 2012 []
Takashima et al. , 2002 []
Efficient selection of the gene conversion event by pac
The drug resistance genes described here can be used simultaneously for multiple transgene expression and gene targeting in mES cells. There is no interference between any of these positive selection markers because we succeeded to obtain mES cells carrying multiple drug-resistance genes in various combinations [20, 27]. Here we described the different kinetics of the drug selection systems pac, zeo, hph, hisD, bsd and neo. Pac and zeo require high levels of expression to confer the proper drug resistance to mES cells, whereas neo establishes the resistance to G418 with minimal expression level in mES cells.
In the conventional transgene expression strategy, the drug resistant genes were driven under the control of strong viral enhancer/promoters derived from simian virus 40 and human cytomegalovirus. However, separation of the expression units of the transgene and the drug-resistant genes failed to ensure the transgene expression in the drug-resistant cells and it was required to screen a large number of drug-resistant clones to obtain the stable transfectants with ideal expression levels. The IRES-mediated bi-cistronic expression vector directs the expression of the transgene and the drug-resistant gene from the same promoter, in which the drug selection always confirms the expression of the transgene. If we apply the rule we identified in this report, it is possible to design an expression vector with an ideal expression level of the transgene by a proper choice of the drug selection system. To obtain the homogenous expression of the fluorescent markers in mES cells, pac and zeo are recommended as we succeeded previously [11, 20]. However, when a moderate or low level of expression is ideal like in the cases of tetracycline-dependent transcriptional activator tTA/rtTA, of which the expression at a high level is toxic for mES cells (Niwa, unpublished), and Tbx3 , neo is the first choice.
We recently reported the function of Esrrb as a target of glycogen synthase kinase-3 (Gsk3)-Tcf3 pathway . In this report we first applied the bi-cistronic expression vector with CAG and IRES-hph to direct the expression of Esrrb in mES cells, resulting in 6–8 fold higher expression of Esrrb transgene than the endogenous levels. Since this situation creates the possibility of neomorphic effects, we switched to the expression vector with IRES-neo and succeeded to confirm that the constitutive expression of the exogenous Esrrb at endogenous levels is sufficient to sustain self-renewal of mES cells in Gsk3 inhibitor-independent manner. This is a good example demonstrating the importance of the choice of a proper drug selection system to obtain appropriate levels of transgene expression. Well-designed strategy for transgene expression will provide clear results in cell biological analyses.
The expression levels of the transgenes using the bi-cistronic expression vectors depend on the drug selection systems. Appropriate choices of the systems will give clean results. This is also applicable to the gene targeting with bi-cistronic durg-resistant genes. The principle shown here in mES cells should be applicable to mouse induced pluripotent stem (iPS) cells directly and most likely to human ES cells after modification.
Initial Methionine of all drug resistance genes in Egfp expression vectors were fused in frame to ATG sequence of the NcoI site in 3′ terminus of EMCV-IRES sequence derived from pCITE-1 (Novagen). pCAG-IP and pCAG-IZ plasmid was constructed for puromycin and zeocin selection as described [9, 28]. pCAG-IB was constructed by replacing the NcoI-XbaI fragment in pCAG-IZ with the BsaI-SpeI fragment containing the bsd gene derived from pUC-SV-BSD (Funakoshi). hisD ORF was amplified from pAGHisD plasmid (a gift from S. Takeda, Kyoto university) by PCR method with sense primer fused to BspHI recognition site and antisense primer fused to XbaI site and exchanged NcoI and XbaI fragment of pGTIRESβgeopA , resulting pGTIRESHisDpA. pCAG-ID was made by replacement of KpnI-BamHI fragment between the pGTIRESHisD and pCAG-IP. PvuI-MscI fragment of pBR322 was ligated to blunted BamHI and PvuI digested pCAG-IP, resulting pBRCAG-IP. pGTIRESHygropA was made by exchanging BspHI-BglII hygro-PGKpA fragment from pSP72-tkphygropA with NcoI-BamHI fragment of pGTIRESβgeopA in which BamHI and BstXI sites within PGKpA are disrupted. pCAG-IH was constructed by exchanging BamHI-KpnI fragment between pBRCAG-IP and pGTIRESHygropA. The puromycin resistance gene of pBRCAG-IP was also exchanged with PCR fragment amplified from pMC1-neo-pA (Stratagene) with BspHI site attached sense primer and both BamHI site and SV40 polyA attached antisense primer, resulting pCAG-IN. pPyCAGIHisDsRedT4 was made by fusing of DsRedT4  open reading frame (ORF) to C-terminus of hisD gene linked with 5′-gagcaagcaagatcgaccaccatg-3′ sequence. The Egfp fragment with XhoI and NotI site obtained from pEGFP-N1 (Clonetech) by PCR was inserted between XhoI and NotI site upstream of IRES in each expression vector backbone, pCAG-IP, -IZ, -ID, -IH, -IB, and –IN, examined for puromycin, zeocin, histidinol, Hygromycin B, blasticisin S, and G418 selection, respectively. pCAG-IpacEGFP was constructed by replacement pac fragment of pCAG-IP with pacEGFP fusion fragment ligated between SalI/NotI digested Egfp fragment from pEGFP-N1 and pac fragment amplified by PCR (sense: 5′-CCTCATGACCGAGTACAAGCCCA-‘3 antisense: 5′-CGGATCCGGCACCGGGCTTGCGGGTCAT-3′) that was digested with BspHI/BamHI, linked between partially filled BamHI and SalI site. hisDsRedT4 ORF was inserted between XhoI and NotI site of pCAG-IP and –IpacEGFP. Full sequence information’s of all expression vectors are available on our web site (http://www.cdb.riken.jp/pcs/protocol/vector/vector_top.html).
Cell culture and electroporation
D3, E14tg2a and EB3 ES cells were maintained in the absence of feeder cells in Glasgow minimal essential medium (GMEM) supplemented with 10% fetal calf serum, 10-4 M 2-mercaptoethanol, and 1000 unit/ml of LIF at normal condition 37°C, 5% CO2 . 2 × 107 ES cells were electroporated with 30 μg of linearized plasmid DNA at 800 V and 3 μF in a 0.4 cm cuvette using a Gene-Pulser (Bio-Rad) and then cultured in the presence of the drugs for selection, Puromycin (Nacalai tesque) Zeocin (Invivogen), Hygromycin B (HygroGold, Invivogen), L-Histidinol (Sigma), Blasticidin S (Invivogen) and G418 (Nacalai tesque), at indicated concentrations. Colonies were identified by Leishman (SIGMA) staining, and counted.
Flow cytometric analysis
Transfectants grown in the presence of each drug concentrations were harvested and 10.000 data points were collected for each sample in flow cytometry, using FACSCALIBUR (Becton Dickinson). Data were analysed using CellQuest Pro Software ver.5.2 (Becton Dickinson).
Gene targeting of Socs3
Genomic DNA sequences were amplified using the primers 5′- ataaatCGatGGCGGCTCTAACTCTGACTCTACACTC-3′ and 5′- ttaagctTGGCGCACGGAGCCAGCGTGGATCTG-3′ (for the left arm of the KO construct); and 5′- CCGGGATcCGGTAGCGGCCGCTGTGCGGAG-3′ and 5′- CAGAGCTCgtcgaCTCCTGTCTGTACAGAAGGAAAGAGAGAG-3′ (for the right arm of the KO construct). Amplified PCR products were cloned into pBR-blue vecotor. The 1.0 kb ClaI (in primer)–NotI (in genomic DNA) fragment from the left arm and the 3.0 kb NotI (in genomic DNA)–SacI (in primer) fragment were cloned into pBR-MC1DTApA. The NotI site was used to clone the marker gene cassette dCherry-IRES-pac-pA. 1 × 107 EB3 ES cells were electroporated with 100 μg of plasmid DNA linealized by XhoI. From the next day, these transfectants were selected with 1.5 μg/ml of puromycin for 8 days. 16 puromycin-resistant colonies were picked, expanded and analyzed their genotype by PCR using the primers 5′- CAGTCCTCCTAGTCGACATTCCTTCTC-3′ 5′- ttaagctTGGCGCACGGAGCCAGCGTGGATCTG-3′ with KOD-Fx (Toyobo) that amplify 2.1 kb fragment from the wild-type allele and 4.8 kb fragment from the targeted allele. One of three homologous recombinants (sKO2) was examined for their ability to survive higher concentration of puromycin, and 1 × 106 sKO2 ES cells were selected with 6 μg/ml of puromycin for 4 days followed by the culture with 1.5 μg/ml of puromycin for 6 days. About 100 colonies were formed and 46 clones were picked, expanded and analyzed their genotype by PCR using the primers shown above.
We thank Dr Futatsugi in our laboratory for editing English. This research was supported by a RIKEN grant.
- Hartman SC, Mulligan RC: Two dominant-acting selectable markers for gene transfer studies in mammalian cells. Proc Natl Acad Sci USA. 1988, 85 (21): 8047-8051. 10.1073/pnas.85.21.8047.View ArticleGoogle Scholar
- Kimura M, Kamakura T, Tao QZ, Kaneko I, Yamaguchi I: Cloning of the blasticidin S deaminase gene (BSD) from Aspergillus terreus and its use as a selectable marker for Schizosaccharomyces pombe and Pyricularia oryzae. Mol Gen Genet. 1994, 242 (2): 121-129. 10.1007/BF00391004.View ArticleGoogle Scholar
- Mulsant P, Gatignol A, Dalens M, Tiraby G: Phleomycin resistance as a dominant selectable marker in CHO cells. Somat Cell Mol Genet. 1988, 14 (3): 243-252. 10.1007/BF01534585.View ArticleGoogle Scholar
- Sugden B, Marsh K, Yates J: A vector that replicates as a plasmid and can be efficiently selected in B-lymphoblasts transformed by Epstein-Barr virus. Mol Cell Biol. 1985, 5 (2): 410-413.View ArticleGoogle Scholar
- Vara JA, Portela A, Ortin J, Jimenez A: Expression in mammalian cells of a gene from Streptomyces alboniger conferring puromycin resistance. Nucleic Acids Res. 1986, 14 (11): 4617-4624. 10.1093/nar/14.11.4617.View ArticleGoogle Scholar
- Yenofsky RL, Fine M, Pellow JW: A mutant neomycin phosphotransferase II gene reduces the resistance of transformants to antibiotic selection pressure. Proc Natl Acad Sci USA. 1990, 87 (9): 3435-3439. 10.1073/pnas.87.9.3435.View ArticleGoogle Scholar
- Reitman M, Lee E, Westphal H, Felsenfeld G: Site-independent expression of the chicken beta A-globin gene in transgenic mice. Nature. 1990, 348 (6303): 749-752. 10.1038/348749a0.View ArticleGoogle Scholar
- Jang SK, Davies MV, Kaufman RJ, Wimmer E: Initiation of protein synthesis by internal entry of ribosomes into the 5′ nontranslated region of encephalomyocarditis virus RNA in vivo. J Virol. 1989, 63 (4): 1651-1660.Google Scholar
- Niwa H, Burdon T, Chambers I, Smith A: Self-renewal of pluripotent embryonic stem cells is mediated via activation of STAT3. Genes Dev. 1998, 12 (13): 2048-2060. 10.1101/gad.12.13.2048.View ArticleGoogle Scholar
- Niwa H, Yamamura K, Miyazaki J: Efficient selection for high-expression transfectants with a novel eukaryotic vector. Gene. 1991, 108 (2): 193-199. 10.1016/0378-1119(91)90434-D.View ArticleGoogle Scholar
- Masui S, Ohtsuka S, Yagi R, Takahashi K, Ko MS, Niwa H: Rex1/Zfp42 is dispensable for pluripotency in mouse ES cells. BMC Dev Biol. 2008, 8: 45-10.1186/1471-213X-8-45.View ArticleGoogle Scholar
- Niwa H, Ogawa K, Shimosato D, Adachi K: A parallel circuit of LIF signalling pathways maintains pluripotency of mouse ES cells. Nature. 2009, 460 (7251): 118-122. 10.1038/nature08113.View ArticleGoogle Scholar
- Tsakiridis A, Tzouanacou E, Larralde O, Watts TM, Wilson V, Forrester L, Brickman JM: A novel fusion reporter system for use in gene trap mutagenesis. Genesis. 2007, 45 (6): 353-360. 10.1002/dvg.20301.View ArticleGoogle Scholar
- Mountford P, Zevnik B, Duwel A, Nichols J, Li M, Dani C, Robertson M, Chambers I, Smith A: Dicistronic targeting constructs: reporters and modifiers of mammalian gene expression. Proc Natl Acad Sci USA. 1994, 91 (10): 4303-4307. 10.1073/pnas.91.10.4303.View ArticleGoogle Scholar
- Gossler A, Joyner AL, Rossant J, Skarnes WC: Mouse embryonic stem cells and reporter constructs to detect developmentally regulated genes. Science. 1989, 244 (4903): 463-465. 10.1126/science.2497519.View ArticleGoogle Scholar
- Skarnes WC, von Melchner H, Wurst W, Hicks G, Nord AS, Cox T, Young SG, Ruiz P, Soriano P, Tessier-Lavigne M, et al: A public gene trap resource for mouse functional genomics. Nat Genet. 2004, 36 (6): 543-544. 10.1038/ng0604-543.View ArticleGoogle Scholar
- Martello G, Sugimoto T, Diamanti E, Joshi A, Hannah R, Ohtsuka S, Gottgens B, Niwa H, Smith A: Esrrb is a pivotal target of the gsk3/tcf3 axis regulating embryonic stem cell self-renewal. Cell Stem Cell. 2012, 11 (4): 491-504. 10.1016/j.stem.2012.06.008.View ArticleGoogle Scholar
- Toyooka Y, Shimosato D, Murakami K, Takahashi K, Niwa H: Identification and characterization of subpopulations in undifferentiated ES cell culture. Development. 2008, 135 (5): 909-918. 10.1242/dev.017400.View ArticleGoogle Scholar
- Nichols J, Zevnik B, Anastassiadis K, Niwa H, Klewe-Nebenius D, Chambers I, Scholer H, Smith A: Formation of pluripotent stem cells in the mammalian embryo depends on the POU transcription factor Oct4. Cell. 1998, 95 (3): 379-391. 10.1016/S0092-8674(00)81769-9.View ArticleGoogle Scholar
- Masui S, Nakatake Y, Toyooka Y, Shimosato D, Yagi R, Takahashi K, Okochi H, Okuda A, Matoba R, Sharov AA, et al: Pluripotency governed by Sox2 via regulation of Oct3/4 expression in mouse embryonic stem cells. Nat Cell Biol. 2007, 9 (6): 625-635. 10.1038/ncb1589.View ArticleGoogle Scholar
- Taniguchi Y, Amazaki M, Furuyama T, Yamaguchi W, Takahara M, Saino O, Wada T, Niwa H, Tashiro F, Miyazaki J, et al: Sema4D deficiency results in an increase in the number of oligodendrocytes in healthy and injured mouse brains. J Neurosci Res. 2009, 87 (13): 2833-2841. 10.1002/jnr.22124.View ArticleGoogle Scholar
- Takashima S, Kitakaze M, Asakura M, Asanuma H, Sanada S, Tashiro F, Niwa H, Miyazaki Ji J, Hirota S, Kitamura Y, et al: Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis. Proc Natl Acad Sci USA. 2002, 99 (6): 3657-3662. 10.1073/pnas.022017899.View ArticleGoogle Scholar
- Vitaterna MH, Selby CP, Todo T, Niwa H, Thompson C, Fruechte EM, Hitomi K, Thresher RJ, Ishikawa T, Miyazaki J, et al: Differential regulation of mammalian period genes and circadian rhythmicity by cryptochromes 1 and 2. Proc Natl Acad Sci USA. 1999, 96 (21): 12114-12119. 10.1073/pnas.96.21.12114.View ArticleGoogle Scholar
- Tateishi S, Niwa H, Miyazaki J, Fujimoto S, Inoue H, Yamaizumi M: Enhanced genomic instability and defective postreplication repair in RAD18 knockout mouse embryonic stem cells. Mol Cell Biol. 2003, 23 (2): 474-481. 10.1128/MCB.23.2.474-481.2003.View ArticleGoogle Scholar
- Nishiyama A, Xin L, Sharov AA, Thomas M, Mowrer G, Meyers E, Piao Y, Metha S, Yee S, Nakatake Y, et al: Uncovering early response of gene regulatory networks in ESCs by systematic induction of transcription factors. Cell Stem Cell. 2009, 5 (4): 420-433.View ArticleGoogle Scholar
- Mortensen RM, Conner DA, Chao S, Geisterfer-Lowrance AA, Seidman JG: Production of homozygous mutant ES cells with a single targeting construct. Mol Cell Biol. 1992, 12 (5): 2391-2395.View ArticleGoogle Scholar
- Niwa H, Miyazaki J, Smith AG: Quantitative expression of Oct-3/4 defines differentiation, dedifferentiation or self-renewal of ES cells. Nat Genet. 2000, 24 (4): 372-376. 10.1038/74199.View ArticleGoogle Scholar
- Niwa H, Masui S, Chambers I, Smith AG, Miyazaki J: Phenotypic complementation establishes requirements for specific POU domain and generic transactivation function of Oct-3/4 in embryonic stem cells. Mol Cell Biol. 2002, 22 (5): 1526-1536. 10.1128/MCB.22.5.1526-1536.2002.View ArticleGoogle Scholar
- Bevis BJ, Glick BS: Rapidly maturing variants of the Discosoma red fluorescent protein (DsRed). Nat Biotechnol. 2002, 20 (1): 83-87. 10.1038/nbt0102-83.View ArticleGoogle Scholar
- Nichols J, Evans EP, Smith AG: Establishment of germ-line-competent embryonic stem (ES) cells using differentiation inhibiting activity. Development. 1990, 110 (4): 1341-1348.Google Scholar
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