pubmed.ncbi.nlm.nih.gov

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells - PubMed

  • ️Fri Jan 01 2010

pEPito: a significantly improved non-viral episomal expression vector for mammalian cells

Rudolf Haase et al. BMC Biotechnol. 2010.

Abstract

Background: The episomal replication of the prototype vector pEPI-1 depends on a transcription unit starting from the constitutively expressed Cytomegalovirus immediate early promoter (CMV-IEP) and directed into a 2000 bp long matrix attachment region sequence (MARS) derived from the human beta-interferon gene. The original pEPI-1 vector contains two mammalian transcription units and a total of 305 CpG islands, which are located predominantly within the vector elements necessary for bacterial propagation and known to be counterproductive for persistent long-term transgene expression.

Results: Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. The pEPito vector is significantly reduced in size, contains only one transcription unit and 60% less CpG motives in comparison to pEPI-1. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. The performance of pEPito-based vectors was further improved by replacing the CMV-IEP with the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element that is known to be less affected by epigenetic silencing events.

Conclusions: The novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.

PubMed Disclaimer

Figures

Figure 1
Figure 1

Mechanism of persistence of MARS-containing pEPI-1 vectors. (A) Episomal replication can be explained by a stable association with early replication foci mediated by the MARS mediated binding to the nuclear matrix protein scaffold attachment factor A (SAF-A). Within early replication foci, the assembly of the origin recognition complex (ORC) and the DNA replication of pEPI vector molecules is likely facilitated by a conformational change resulting from mRNA transcription.(B) Nuclear retention and mitotic stability of pEPI-based vectors can be explained by MARS mediated direct or indirect interaction with metaphase chromosomes in a "piggy back"-like mechanism. (C) The functional element of pEPI-vector plasmid replicons consists of a transcription unit, regulated by a constitutive promoter and directed into a chromosomal MARS with the prerequisite of no termination signal being located between transcription unit and MARS element.

Figure 2
Figure 2

pEPI-derived plasmids generated in this study. A schematic picture of the four types of vector backbones is depicted in the upper panel: pEPI-1 (A), pEPito (B), pEPI-1-ΔMARS (C), or pEPito-ΔMARS (D). All vectors contain either the CMV-IEP or the hCMV/EF1P promoter element. Depending on the experiment, three different transgenes have been used: an EGFP-BSD cassette connected via an internal ribosomal entry site module (EGFP-IRES-BSD), a Firefly luciferase (Luc), or an EGFP-luciferase fusion protein (EGFP::Luc). All 13 different vectors have been constructed, propagated, and amplified in E.coli DB3.1λpir. (E) Additional information regarding the different vectors. The vector pEPI-1- [CMV-IEP]- [Luc] has been recently published as pEPI-Luc [32]. This figure does not claim proportional correctness.

Figure 3
Figure 3

Transfection efficiencies and expression levels in transiently transfected HEK293 and NIH3T3 cells. Transient transfection efficiencies (A, B) and the transient mean EGFP expression levels per cell (C, D) obtained by transfection of the vectors #1-8 into HEK293 cells (A, C), or NIH3T3 cells (B, D). All vectors contain the EGFP-IRES-BSD transgene transcription unit. Mean values of n = 8 derived from four independent experiments ± SD are shown; * p < 0.05; ** p < 0.001 (two-tailed Student's t-test). mock: untransfected control. Grey bars indicate vectors containing an hCMV/EF1P promoter element, black bars indicate vectors containing a CMV-IEP promoter element.

Figure 4
Figure 4

Expression levels and colony formation efficiencies in stably selected HEK293 and NIH3T3 cells. (A, B) Mean EGFP expression levels per cell (A: HEK293, B: NIH3T3) of stably selected cells. (C, D) Relative colony-forming efficiency of stably selected HEK293 cells (C, normalized to pEPI-1- [CMV-IEP]- [EGFP-IRES-BSD]-ΔMARS) or NIH3T3 cells (B, normalized to pEPI-1- [hCMV/EF1P]- [EGFP-IRES-BSD]-ΔMARS. All vectors contain the EGFP-IRES-BSD transgene transcription unit. Mean values of n = 8 derived from four independent experiments ± SD are shown; * p < 0.05; ** p < 0.001 (two-tailed Student's t-test). mock: untransfected control. n.d.: no stable mixed-clones obtained after transfection and blasticidin selection. Grey bars: vectors containing the hCMV/EF1 promoter element; black bars: vectors containing the CMV-IEP promoter element.

Figure 5
Figure 5

Increased and prolonged expression of pEPito in vivo. Luciferase expression profiles of exemplary MF-1 mice hydrodynamically injected with the vectors #9-13 (Figure 2 as assayed by in vivo bioluminescence imaging after 1 (first column), 7 (second column), 14 (third column), and 32 (fourth column) days post injection. The vector pEPI-1- [CMV-IEP]- [Luc] has been previously published as pEPI-Luc [32].

Similar articles

Cited by

References

    1. Piechaczek C, Fetzer C, Baiker A, Bode J, Lipps HJ. A vector based on the SV40 origin of replication and chromosomal S/MARs replicates episomally in CHO cells. Nucleic Acids Res. 1999;27:426–428. doi: 10.1093/nar/27.2.426. - DOI - PMC - PubMed
    1. Bode J, Kohwi Y, Dickinson L, Joh T, Klehr D, Mielke C, Kohwi-Shigematsu T. Biological significance of unwinding capability of nuclear matrix-associating DNAs. Science. 1992;255:195–197. doi: 10.1126/science.1553545. - DOI - PubMed
    1. Baiker A, Maercker C, Piechaczek C, Schmidt SB, Bode J, Benham C, Lipps HJ. Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix. Nat Cell Biol. 2000;2:182–184. doi: 10.1038/35004061. - DOI - PubMed
    1. Jenke AC, Stehle IM, Herrmann F, Eisenberger T, Baiker A, Bode J, Fackelmayer FO, Lipps HJ. Nuclear scaffold/matrix attached region modules linked to a transcription unit are sufficient for replication and maintenance of a mammalian episome. Proc Natl Acad Sci USA. 2004;101:11322–11327. doi: 10.1073/pnas.0401355101. - DOI - PMC - PubMed
    1. Jenke AC, Eisenberger T, Baiker A, Stehle IM, Wirth S, Lipps HJ. The nonviral episomal replicating vector pEPI-1 allows long-term inhibition of bcr-abl expression by shRNA. Hum Gene Ther. 2005;16:533–539. doi: 10.1089/hum.2005.16.533. - DOI - PubMed

Publication types

MeSH terms

LinkOut - more resources