Abstract
BackgroundThe 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 β-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.ResultsHere, 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.ConclusionsThe novel vector pEPito can be considered suitable as an improved vector for biotechnological applications in vitro and for non-viral gene delivery in vivo.
Highlights
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 β-interferon gene
The non-viral plasmid vector pEPI-1 was constructed in 1999 by Piechaczek et al [1], by cloning a 2000 bp long matrix attachment region sequence (MARS), that was derived of the human β-interferon gene cluster [2], into the commercial vector pGFP-C1 (Clontech, USA)
Generation of pEPI-based vectors with improved efficacy We systematically generated a panel of 13 different pEPI1-derived vectors, all of them constructed in a modular way enabling the easy exchange of backbone, promoter, or transgene elements by restriction digest with PciI, NheI, BglII, or MluI (Figure 2)
Summary
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 β-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. The first, Cytomegalovirus immediate early promoter (CMV-IEP) driven enhanced green fluorescent protein (EGFP) transcription unit is oriented into the MARS and has been shown to be a functional component of the pEPI-1 vector plasmid replicon. This is the case when pEPI-1 plasmids are isolated from already established mammalian cell clones and reintroduced into cells demonstrating that the primary sequence of DNA is not involved in the establishment process [8,11,12] but rather epigenetic features such as chromatin structure and nuclear localization [8]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
