pCpGfree-basic

PLASMID FEATURES

All the elements required for replication and selection of the plasmid in E. coli and gene expression in mammalian cells are completely devoid of CpG dinucleotides. Furthermore, all Dam methylation sites (GATC) have been removed to prevent prokaryotic methylation.

Elements for expression in E. coli

• Origin of replication: The E. coli R6K gamma ori has been modified to remove all CpGs. This origin is activated by the R6K specific initiator protein π, encoded by the pir gene [1].
• Bacterial promoter: EM2K is a CpG-free version of the bacterial EM7 promoter.
• Selectable marker: The Zeocin® resistance gene is a small gene (<400 bp) that contains numerous CpG dinucleotides. A synthetic new allele was created that contains no CpGs.

Elements for expression in mammalian cells

• The synthetic mSEAP∆CpG gene: a CpG-free allele of the murine SEAP gene constructed by chemical synthesis.
• Polyadenylation signal: The polyadenylation signal is a CpG-free form of the late SV40 polyadenylation signal.
• MAR: Matrix attached regions (MARs) are sequences typically AT-rich that are able to form barriers between independently regulated domains [2]. pCpGfree plasmids contains two MARs, from the 5’ region of the human IFN-β gene or β-globin gene that were chosen because they are naturally CpG-free. The MARs are placed between the bacterial and mammalian transcription units.
• MCS: The multiple cloning site contains several commonly used restriction sites for convenient cloning of a gene of interest.    5’ Sda I, Bsp 120I,Avr II, Nsi I, Ppu 10I, Sca I, Bam HI, Spe I 3’

Due to the presence of the R6Kγ origin of replication, pCpGfree-basic can only be amplified in E. coli mutant strain expressing a pir mutant gene. It will not replicate in standard E. coli strains. Therefore, pCpGfree-basic is provided with the E. coli GT115 strain, a pir mutant also deficient in Dcm methylation.

1.  Wu F. et al. 1995. A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J Bacteriol. 177(22):6338-45.
2. Bode J. et al., 1996. Scaffold/matrix-attached regions: topological switches with multiple regulatory functions. Crit Rev Eukaryot Gene Expr. 6(2-3):115-38.

Procedure

1- Insert promoter and/or enhancer fragment into pCpGfree-basic
2- Treat recombinant plasmid with CpG methylase (Sss I) or site specific-methylase (Hha I, Hpa II).
3- Transiently transfect cells with methylase-treated or untreated plasmid.
4- Analyze promoter CpG methylation by determining reporter expression using the appropriate detection reagent.