pCpGfree-siRNA is a plasmid that combines the backbone of the pCpG plasmids with the siRNA expression cassette of the psiRNA plasmids. It is designed for long lasting expression of shRNAs in vivo as the plasmid does not induce inflammatory responses and gene silencing by methylation in vertebrate hosts.
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Description
CpG-free Plasmid Backbone 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.
Enhanced RNA Polymerase III Promoter pCpGfree-siRNA plasmid features the hum>n 7SK promoter, in which all CpG dinucleotides have been removed. The activity of this RNA Pol,III promoter is augmented by the addition of the enhancer of the CMV immediate-early promoter, a Pol II enhancer [1]. The enhancer has been modified to remove all CpGs.
Easy and Convenient Cloning of the siRNA Insert Cloning options: pCpGfree-siRNA plasmid uses Bbs I, an unusual restriction enzyme that generates asymmetric cohesive overhangs that are not compatible with each other to eliminate the risk of self-ligation of the vector. A second cloning strategy can be chosen by using Acc 65I and Hind III restriction sites. White/blue selection: pCpGfree-siRNA plasmids exploit the white/blue selection system to facilitate the screening of recombinant clones. They feature an EM7-LacZ α-peptide cassette between the cloning sites to allow the easy discrimination between blue parental and white recombinant clones.
1. Xia XG. et al. 2003. An enhanced U6 promoter for synthesis of short hairpin RNA. Nucleic Acids Res. 31(17):e100. 2. Wu F. et al. 1995. A DNA segment conferring stable maintenance on R6K gamma-origin core replicons. J Bacteriol. 177(22):6338-45. 3. Connelly JC. et al., 1998. The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA. PNAS USA. 95(14):7969-74.
Contents and Storage
pCpGfree-siRNA plasmid is provided in a kit that includes the following components: - 50 μg pCpGfree-siRNA plasmid - 20 μg control pCpGfree-siRNA plasmid expressing a scramble sequence - 1 vial of LyoComp GT115 - 10 μg of each sequencing primer - 4 pouches of Fast-Media® Zeo X-Gal Plasmids are provided as lyophilized DNA. Kit is shipped at room temperature. Store Fast-Media® pouches at room temperature. Store all other components at -20°C.
Important Licencing Information
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Note that the sequence of available ORFs provided by InvivoGen can differ from a given reference Genbank record due to genetic variations
and/or alternative splicing. Customers should verify that the version of a gene sold by InvivoGen is suitable for the customer needs.
