Simultaneous knockdown of the expression of two genes using multiple shRNAs and subsequent knock-in of their expression.
Small hairpin RNA (shRNA) is a powerful tool for inhibiting gene expression. One limitation has been that this technique has been used primarily to target a single gene. This protocol expands upon previous methods by describing a knockdown vector that facilitates cloning of multiple shRNAs; this allows targeted knockdown of more than one gene or of a single gene that may otherwise be difficult to knockdown using a single shRNA. The targeted gene(s) can be readily re-expressed by transfecting knockdown cells with a knock-in vector, containing an shRNA-refractive cDNA that will express the protein-of-interest even in the presence of shRNAs. The constructed knockdown and knock-in vectors can be easily used concurrently to assess possible interrelationships between genes, the effects of gene loss on cell function and/or their restoration by replacing targeted genes one at a time. The entire knockdown or knock-in procedure can be completed in approximately 3-4 months.
Xu
Xue-Ming
XM
Molecular Biology of Selenium Section, Laboratory of Cancer Prevention, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, Maryland, USA.
Yoo
Min-Hyuk
MH
Carlson
Bradley A
BA
Gladyshev
Vadim N
VN
Hatfield
Dolph L
DL
D015870:Gene Expression; D055879:Gene Knock-In Techniques; D055786:Gene Knockout Techniques; D005822:Genetic Vectors; D016296:Mutagenesis; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000737:chemistry; D022661:RNA, Untranslated / Q000737:chemistry; D014162:Transfection / Q000379:methods*
D034741:RNA, Small Interfering; D022661:RNA, Untranslated