Robust Expression of Transgenes in MCF-7 Breast Cancer Cells is Expression Vector-Dependent (2004)

BioTechniquesVol. 37, No. 3 BenchmarksOpen AccessRobust expression of transgenes in MCF-7 breast cancer cells is expression vector-dependentPaul Winnard, Yelena Mironchik & Venu RamanPaul WinnardThe Johns Hopkins University, Baltimore, MD, USA, Yelena MironchikThe Johns Hopkins University, Baltimore, MD, USA & Venu Raman*Address correspondence to: Venu Raman, The Johns Hopkins University, Dept of Radiology, 340 Traylor, 720 Rutland Avenue, Baltimore, MD 21205, USA. e-mail: E-mail Address: vraman2@jhmi.eduThe Johns Hopkins University, Baltimore, MD, USAPublished Online:6 Jun 2018https://doi.org/10.2144/04373BM06AboutSectionsPDF/EPUB ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinkedInRedditEmail We are reporting that the in vivo protein expression in mammalian MCF-7 breast cancer cells can be highly dependent on the type of commercially available expression vectors used. Figure 1 is a representation of the vector backbones used and indicates the position of our genes of interest (denoted Insert) relative to the cytomegalovirus (CMV) promoter and simian virus 40 (SV40) poly(A) signal sequence in a pCMVTag4A vector (Stratagene, La Jolla, CA, USA), which resulted in an in-frame fusion at the 3′ end with a FLAG® epitope sequence. Figure 1 also depicts the placement of these FLAG-tagged genes (including a TGA stop codon) into a pEF-1α-His/Myc vector (Invitrogen, Carlsbad, CA, USA) and their relative positions with respect to the elongation factor-1α (EF-1α) promoter and bovine growth hormone (BGH) poly(A). Thus, except for the vector backbones, both fusion gene construct sequences were identical in the two vector systems. Sequencing analyses proved that each gene sequence was correct and in-frame with the FLAG sequence and stop codon. All plasmids were prepared using the Qiagen® Plasmid Midi Kits (Qiagen, Valencia, CA, USA). Initial transient transfection experiments indicated that little or no transgene-encoded proteins were made in MCF-7 cells transfected with the Stratagene vector constructs. In order to eliminate the possibility that the Stratagene-based plasmid preparations were contaminated with an inhibitory substance(s) or had some mutations, we performed a second round of experiments. Stratagene-based plasmid constructs were grown, put through two rounds of plasmid purification (260:280 = 1.75 to 1.80), and once again sequenced, which eliminated the questions about any acquired mutations. In order to semiquantitatively evaluate transgene expression, transient transfection experiments included either the Renilla luciferase or enhanced green fluorescent protein (EGFP) reporter expression vectors. Figure 2 shows a representative immunoblot (out of three that were semiquantitative blots and seven nonquantified blots). Figure 2 indicates that EF-1α-BGH p(A) vector constructs provide a sequence context, which in MCF-7 cells, drives considerably higher transgene expression (lanes 1 and 3) than the CMVSV40 p(A) vector constructs (lanes 2 and 4) within our experimental conditions. Protein bands seen in lanes 2 and 4 of Figure 2 only become visible after prolonged exposure (30 min), which is an indication of the low expression of the FLAG-tagged proteins in cells transfected with the Stratagene CMV promoter-SV40 p(A)-based vector constructs. In contrast, 10 s exposures (data not shown) were more than adequate for the visualization of proteins driven of the EF-1α-based vectors. These results indicate that in MCF-7 breast cancer cells, transgene expression can be highly dependent upon the expression vector used. However, in order to obtain a better understanding of the vector component(s) that may be primarily responsible for the enhanced expression seen with the EF-1α-based vectors, we replaced the EF-1α promoter in one of our EF-1α vectors with the CMV promoter from pCMVTag4A-Flag for use in another round of transient transfection experiments. Figure 3 illustrates that the CMV promoter can generate protein expression levels that are only about two times less than those generated by the EF-1α promoter when it is within the context of the EF-1α-based vector backbone. Taken together, these results indicate that vector-driven in vivo protein expression in MCF-7 cells can be vector-dependent. The cases tested here, Invitrogen's pEF-1α-His/Myc vector versus Stratagene's pCMVTag4A vector, show that the majority of the loss of protein expression from pCMVTag4A-based constructs can be attributed to vector components or an arrangement of vector components in Stratagene's vector backbone other than its promoter. However, under the conditions used within this study, the EF-1α promoter consistently produced higher levels of protein.Figure 1. Illustration of the mammalian expression vector constructs used in this study.Locations of each vector's major regulatory components along with the genes of choice (labeled Insert) are depicted. Differences include promoters and polyadenylation signal sequences as well as the placement of the ColE1 ori sequences and vector size. Genes were inserted into Stratagene's pCMVTag4A vector in-frame with a FLAG epitope sequence. FLAG-tagged genes along with TGA stop codons were excised from the pCMVTag4A-Flag vectors with SacII and KpnI digestion, blunt-ended (T4 DNA polymerase), and ligated into the EcoRV site of Invitrogen's pEF-1α-His/Myc vector, creating the pEF-1α-Flag-stop constructs. MCS, multiple cloning site; CMV, cytomegalovirus; TK, thymidine kinase; EF-1α, elongation factor-1α; SV40, simian virus 40; pA, polyadenylation; BGH, bovine growth hormone.Figure 2. Representative immunoblot indicating protein expression levels in cell lysates from transiently transfected MCF-7 cells.Lanes 1 and 3 indicate the large amounts of protein expression obtained from pEF-1α-His/Myc vector constructs as compared to the low levels of protein expression (lanes 2 and 4) obtained from the pCMVTag4A vector construct. Two different FLAG-tagged proteins are shown: lanes 1 and 2 (Insert 1) versus lanes 3 and 4 (Insert 2).Figure 3. Representative immunoblot comparing protein expression levels from pEF-1α-His/Myc vector constructs in cell lysates from transiently transfected MCF-7 cells.The right-hand lane shows protein expression from one of our pEF-1α-Flag-Stop vector constructs. The left-hand lane shows protein expression from a vector prepared from the same pEF-1α-Flag-Stop vector construct where the elongation factor-1α (EF-1α) promoter has been replaced by the cytomegalovirus (CMV) promoter. This blot indicates that, even within the context of an identical vector backbone, the EF-1α promoter gives higher protein expression (approximately 2-fold higher; n = 2) than the CMV promoter. Protein loading was normalized to Renilla luciferase luminescence.A variety of reasons could explain these results although most have not been systematically studied by us. Mammalian expression vector development (1,2) has gone hand in hand with a continuous effort to increase the efficacy of these vectors (3–6). Optimal promoter activity has often been the focus of such design efforts (e.g., Reference 5). Throughout continued testing, the CMV promoter has been proven to give robust in vivo expression of an investigator's choice of transgenes (7,8). However, certain limitations with the use of the CMV promoter have also been reported. For example, it has been shown that the CMV promoter can be silenced (9). In addition, the CMV promoter can be down-regulated by wild-type p53 tumor suppressor protein (10). An alternative promoter that has come into routine use is the EF-1α promoter (11–13). However, comparisons of the capabilities of the CMV versus the EF-1α promoter to drive expression of mammalian trans-genes have produced equivocal results (e.g., compare Reference 8 and Reference 13). For example, Kim et al. (14) have recently reported that generally the EF-1α promoter is superior in driving transgene expression as compared to the CMV promoter due to the inclusion of the first intron of the EF-1α gene within the context of the promoter. Evidence is also building that the EF-1α promoter may have advantages over the CMV promoter with respect to persistent transgene expression in stable clones (11,13). Another component of vector engineering that has come under study has been the choice of poly(A) signal sequence (15,16). Yet, as with promoter comparisons, comparisons of the SV40 poly(A) sequence versus the BGH poly(A) sequence can produce equivocal results (15,16). These reports and others (e.g., Reference 17) indicate that many factors can contribute to the regulation of transgene expression in mammalian cells. Thus, it is prudent to analyze more than one promoter and backbone sequence for its ability to modulate high protein expression within a given cell type or tissue. This is important if an efficient evaluation of the overexpressed protein's in vivo function(s) as well as its impact on cellular physiology is to be made. On the other hand, vector-driven synthesis of suboptimal or nondetectable quantities of protein makes an evaluation of the protein's effect(s) on cellular function a dubious and expensive task. Such considerations may be particularly pertinent in the context of cancer cells, such as MCF-7s, because dysregulation of genetic information is the norm in such cells and is likely to affect protein synthesis.AcknowledgmentsThis work was supported by National Institutes of Health grant no. P50 CA103175 to V.R.Competing Interests StatementThe authors declare that they have no competing interestsReferences1. Mulligan, R.B., B. Howard, and P. Berg. 1979. Synthesis of rabbit β-globin in cultured monkey cells following infection with a SV40 β-globin recombinant genome. Nature 227:108–114.Crossref, Google Scholar2. Gorman, C.M., L.F. Moffat, and B.H Howard. 1982. Recombinant genomes which express chloramphicol acetyltransferase in mammalian cells. Mol. Cell. Biol. 2:1044–1051.Crossref, Medline, CAS, Google Scholar3. Takebe, Y., M. Seiki, J.-I. Fujisawa, P. Hoy, K. Yokota, K.-I. Arai, M. Yoshida, and N. Arai. 1988. SRα promoter: an efficient and versatile mammalian cDNA expression system composed of the simian virus 40 early promoter and the RU5 segment of human T-cell leukemia virus type 1 long terminal repeat. Mol. Cell. Biol. 8:466–472.Crossref, Medline, CAS, Google Scholar4. Ray, J. and F.H. Gage. 1992. Gene transfer into established and primary fibroblast cell lines: comparison of transfection methods and promoters. BioTechniques 12:598–603.Google Scholar5. Wenger, R.H., H. Moreau, and P.J. Nielsen. 1994. A comparison of different promoter, enhancer, and cell type combinations in transient transfections. Anal. Biochem. 221:416–418.Crossref, Medline, CAS, Google Scholar6. Kaufman, R.J. 2000. Overview of vector design for mammalian gene expression. Mol. Biotechnol. 16:151–160.Crossref, Medline, CAS, Google Scholar7. Foecking, M.K. and H. Hofstetter. 1986. Powerful and versatile enhancer-promoter unit for mammalian expression vectors. Gene 45:101–105.Crossref, Medline, CAS, Google Scholar8. Lipshutz, G.S., D. Titre, M. Brindle, A.R. Bisconte, C.H. Contag, and K.M.L. Gaensler. 2003. Comparison of gene expression after intraperitoneal delivery of AAV2 or AAV5 in utero. Mol. Ther. 8:90–98.Crossref, Medline, CAS, Google Scholar9. Loser, P., G.S. Jennings, M. Strauss, and V. Sandig. 1998. Reactivation of the previously silenced cytomegalovirus major immediate-early promoter in the mouse liver: involvement of NFκB. J. Virol. 72:180–190.Crossref, Medline, CAS, Google Scholar10. Allamane, S., D. Ratel, P. Jourdes, F. Berger, A.L. Benabid, and D. Wion. 2001. p53 status and gene transfer experiments using CMV enhancer/promoter. Biochem. Biophys. Res. Commun. 280:45–47.Crossref, Medline, CAS, Google Scholar11. Najjar, S.M. and R.E. Lewis. 1999. Persistent expression of foreign genes in cultured hepatocytes: expression vectors. Gene 230:41–45.Crossref, Medline, CAS, Google Scholar12. Kim, D.W., T. Uetsuki, Y. Kaziro, N. Yamaguchi, and S. Sugano. 1990. Use of the human elongation factor 1 alpha promoter as a versatile and efficient expression system. Gene 91:217–223.Crossref, Medline, CAS, Google Scholar13. Chung, S., T. Andersson, K.-C. Sonntag, L. Bjorklund, O. Isacson, and K.-S. Kim. 2002. Analysis of different promoter systems for efficient transgene expression in mouse embryonic stem cell lines. Stem Cells 20:139–145.Crossref, Medline, CAS, Google Scholar14. Kim, S.-Y., J.-H. Lee, H.-S. Shin, H.-J. Kang, and Y.-S. Kim. 2002. The human elongation factor 1 alpha (EF-1α) first intron highly enhances expression of foreign genes from the murine cytomegalovirus promoter. J. Biotechnol. 93:183–187.Crossref, Medline, CAS, Google Scholar15. Xu, Z.-L., H. Mizuguchi, A. Ishii-Watabe, E. Uchida, T. Mayumi, and T. Hayakawa. 2001. Optimization of transcriptional regulatory elements for constructing plasmid vectors. Gene 272:149–156.Crossref, Medline, CAS, Google Scholar16. Xu, Z.-L., H. Mizuguchi, A. Ishii-Watabe, E. Uchida, T. Mayumi, and T. Hayakawa. 2002. Strength evaluation of transcriptional regulatory elements for transgene expression by adenovirus vector. J. Control Release 81:155–163.Crossref, Medline, CAS, Google Scholar17. Chen, Z.-Y., C.-Y. He, A. Ehrhardt, and M.A. Kay. 2003. Minicircle DNA vectors devoid of bacterial DNA result in persistent and high-level transgene expression in vivo. Mol. Ther. 8:495–500.Crossref, Medline, CAS, Google ScholarFiguresReferencesRelatedDetailsCited ByDeveloping MR reporter genes: promises and pitfalls1 January 2007 | NMR in Biomedicine, Vol. 20, No. 3Magnetic resonance imaging and spectroscopy of transgenic models of cancer1 January 2007 | NMR in Biomedicine, Vol. 20, No. 3 Vol. 37, No. 3 Follow us on social media for the latest updates Metrics Downloaded 737 times History Received 17 February 2004 Accepted 12 May 2004 Published online 6 June 2018 Published in print September 2004 Information© 2004 Author(s)AcknowledgmentsThis work was supported by National Institutes of Health grant no. P50 CA103175 to V.R.Competing Interests StatementThe authors declare that they have no competing interestsPDF download