Construction of Modular Lentiviral Vectors for Effective Gene Expression and Knockdown.
Elucidating gene function is heavily reliant on the ability to modulate gene expression in biological model systems. Although transient expression systems can provide useful information about the biological outcome resulting from short-term gene overexpression or silencing, methods providing stable integration of desired expression constructs (cDNA or RNA interference) are often preferred for functional studies. To this end, lentiviral vectors offer the ability to deliver long-term and regulated gene expression to mammalian cells, including the expression of gene targeting small hairpin RNAs (shRNAmirs). Unfortunately, constructing vectors containing the desired combination of cDNAs, markers, and shRNAmirs can be cumbersome and time-consuming if using traditional sequence based restriction enzyme and ligation-dependent methods. Here we describe the use of a recombination based Gateway cloning strategy to rapidly and efficiently produce recombinant lentiviral vectors for the expression of one or more cDNAs with or without simultaneous shRNAmir expression. Additionally, we describe a luciferase-based approach to rapidly triage shRNAs for knockdown efficacy and specificity without the need to create stable shRNAmir expressing cells.
Department of Biology, McGill University, Montréal, QC, H3G 0B1, Canada.
Angeline
A
de Bruyns
Department of Biology, McGill University, Montréal, QC, H3G 0B1, Canada.
Ben
B
Geiling
Department of Biology, McGill University, Montréal, QC, H3G 0B1, Canada. david.dankort@mcgill.ca.
David
D
Dankort
D018076:DNA, Complementary / Q000235:genetics*; D015870:Gene Expression / Q000235:genetics; D055785:Gene Knockdown Techniques / Q000379:methods*; D005822:Genetic Vectors; D006801:Humans; D016086:Lentivirus / Q000235:genetics*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000235:genetics
D018076:DNA, Complementary; D034741:RNA, Small Interfering
Delivery; Design; Dual-luciferase reporter; Gateway cloning; Lentiviral vectors; Lentivirus; Short hairpin RNA (shRNA); shRNA triage