A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome
Dubravka Skalamera, Max V Ranall, Benjamin M Wilson, Paul Leo, Amy S Purdon, Carolyn Hyde, Ehsan Nourbakhsh, Sean M Grimmond, Simon C Barry, Brian Gabrielli, Thomas J Gonda
PLoS One | PUBLIC LIBRARY SCIENCE | Published : 2011
In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T..View full abstract
Awarded by National Health and Medical Research Council of Australia (NHMRC)
Awarded by Australia Research Council (ARC)
This work was supported by funding from the National Health and Medical Research Council of Australia (NHMRC, http://www.nhmrc.gov.au/; grant ID401608), the Australia Research Council (ARC, http://www.arc.gov.au/; grant LE0668241) and the Australian Cancer Research Foundation (ACRF, http://www.acrf.com.au/). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.