Overexpression is the New Alternative
While conceptually simpler than RNAi knockdown, cDNA overexpression is very much the newcomer to genome-scale screening. One obvious advantage is that a cDNA only overexpresses the one transcript that it encodes; the sequence-based off-target effects that afflict sub-optimal RNAi designs are not possible. Moreover, levels of an overexpressed protein can be many times higher than normal, which has the potential to alter the phenotype dramatically.
This may expand the dynamic range of the readout, increasing the sensitivity of the screen. (On the other hand, high concentrations of an overexpressed protein might be toxic to the cell or may increase the probability of low-affinity binding interactions, stimulating off-target biochemical pathways.) However, cDNA overexpression screens are limited by the availability of fully sequenced, full-ORF-containing cDNAs cloned into a mammalian expression vector.
The gold standard Mammalian Gene Collection (MGC) contains fully sequenced cDNAs verified to contain a complete coding sequence. A subset of this collection has been cloned into the mammalian expression vector pCMV-SPORT6 and contains 6,415 human cDNAs and 9,624 mouse cDNAs. This subset of MGC cDNAs (including human and mouse) has been employed in highly fruitful cDNA overexpression screens with human cell lines. It is currently the largest collection of full-length, expression-ready cDNAs and is available from Open Biosystems as lyophilized DNA in 384-well screening plates (Assay-Ready MGC cDNA), as normalized aqueous solutions of DNA, or as glycerol stocks.
Until now, cDNA overexpression has lagged behind RNAi in the range of vectors employed for delivery and the use of pooled screening formats. However, there is nothing intrinsic to the technology that prevents the use of these enhancements. The content available for genome-scale overexpression screening has also lagged behind RNAi, though with the continued growth of the MGC, it will gradually catch up.