Abstract:
:RNA interference is a widely used tool for analysis of gene function in mammalian cells. Stable knockdown of specific target genes can be maintained in cell lines and live organisms using vector-based delivery of short hairpins (shRNAs) driven by RNA polymerase III promoters. Here we describe a vector incorporating the human 7SK promoter for shRNA-mediated gene silencing in the P19 embryonic carcinoma stem cell line. Our preliminary experiments with the 7SK shRNA expression vector indicated that its activity could be hindered by random genomic integration. In order to counter this inhibitory mechanism, we inserted a matrix-attached region sequence to generate an episomal vector system. We compared the effects of insertion versus exclusion of the MAR sequence on the shRNA-mediated gene-specific silencing of the beta-tubulin III and Cyclophilin A genes. While the MAR sequence is not strongly correlated with the episomal status of the expression vector, our studies indicate that inclusion of the MAR element significantly enhances the stability of shRNA-mediated gene silencing in the P19 stem cells.
journal_name
Plasmidjournal_title
Plasmidauthors
Sotirova VN,Calciano MA,Krueger W,Lalande Mdoi
10.1016/j.plasmid.2005.11.008keywords:
subject
Has Abstractpub_date
2006-05-01 00:00:00pages
216-26issue
3eissn
0147-619Xissn
1095-9890pii
S0147-619X(05)00125-3journal_volume
55pub_type
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