Efficient downregulation of multiple mRNA targets with a single shRNA-expressing lentiviral vector.
Gene silencing based on RNA interference is widely used in fundamental research and in practical applications. However, a commonly incomplete functional suppression represents a serious drawback of this technology. We describe a series of lentiviral vectors each containing a single or multiple shRNA-expression cassette(s) driven by a RNA-polymerase III specific promoter and localized within the 3'-LTR of the lentiviral DNA backbone. The vectors also contain an antibiotic-resistance gene that allows positive selection of recipient cells. The combined expression of three different shRNAs specific to a single mRNA was shown to improve dramatically the level of mRNA inhibition, while the use of three different RNA-polymerase III specific promoters avoids the loss of shRNA-expression cassettes through the homologous recombination. The vector system was used for successful simultaneous suppression of three related SESN1, SESN2 and SESN3 genes, which suggests its particular value for testing phenotypes of functionally redundant genes.
Chumakov
Stepan P
SP
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Street 32, 119991 Moscow, Russia.
Kravchenko
Julia E
JE
Prassolov
Vladimir S
VS
Frolova
Elena I
EI
Chumakov
Peter M
PM
D002460:Cell Line; D015536:Down-Regulation / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics*; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D012320:RNA Polymerase III / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000378:metabolism*; D020079:Terminal Repeat Sequences / Q000235:genetics
D012333:RNA, Messenger; D034741:RNA, Small Interfering; D012320:RNA Polymerase III