There are now several approaches to predicting gene functions on a genome scale, including bioinformatics, gene expression microarrays, and yeast two-hybrid screens. Cell-based assays are particularly useful in that they enable the discovery of causal connections between gene activity and a cellular phenotype of interest. The most popular tool for modulating gene activity is RNAi knockdown, but the logical alternative, cDNA overexpression, has also proven fruitful in discovering new gene functions. Why, then, has it gotten so little attention compared to RNAi? Let's see how these two alternatives actually compare.
It is a simple fact that there are more RefSeq mRNA sequences known than there are full-length cDNA clones available. There are 18,000 genes represented in human RefSeq, but only 13,500 are represented in the Mammalian Gene Collection (MGC) and less than 6000 of these are in the most common mammalian expression vector, pCMVSPORT6. All 18,000 RefSeq-defined genes can be targeted for RNAi knockdown. Advantage: RNAi.
The rules governing the efficiency of RNAi knockdown, whether using siRNA, shRNA or shRNAmir triggers, are still being learned and successful designs must be discovered by trial and error. For cDNA overexpression, nature has already provided the design. Advantage: cDNA.
SEQUENCE-SPECIFIC OFF-TARGET EFFECTS
RNAi triggers must be designed to hybridize with one target mRNA sequence and not with other similar sequences in the transcriptome. This is not easy. Splice variants represent a particularly difficult design challenge for microarray probes—and for RNAi. Do you target each gene isoform individually or together using one common design? Neither goal may be achievable in practice, especially given that the entire catalog of isoforms may not be known for many years to come. By contrast, each cDNA codes for exactly one mRNA and exactly one protein. It doesn't get any more specific than that. Advantage: cDNA.
OTHER OFF-TARGET EFFECTS
While transfecting siRNAs may lead to cytotoxicity or an interferon response, these off-target effects seem to be largely avoided using shRNAmir technology. On the other hand, high concentrations of an overexpressed protein may be cytotoxic or may increase the probability of low-affinity binding interactions that stimulate off-target biochemical pathways. Advantage: RNAi.
RNAi triggers are short and of uniform length, so deliverability into cells will be consistently high. While transfection efficiency for cDNA is largely independent of length, the field of RNAi has demonstrated the advantages of viral vectors, where packaging may limit deliverability of the longest cDNAs. Advantage: RNAi.
A protein target can never be knocked down by more than 100%, which is the maximum possible dynamic range for RNAi. On the other hand, a protein target may be overexpressed by more than 100% and, possibly, many hundreds of percent. This greater dynamic range may increase the sensitivity of an assay in some cases. Advantage: cDNA.
ONE OR THE OTHER—OR BOTH?
Where is all this heading? For the foreseeable future, RNAi collections will be able to offer broader gene coverage, especially for longer genes. However, the MGC will continue to grow and, as gene synthesis becomes more economical, may begin to close the coverage gap. Overexpression has an intrinsic and (very likely) enduring advantage in specificity.
As researchers become more interested in distinguishing between the functions of alternatively spliced transcripts of the same gene, this advantage will become more important. Overexpression screens may also prove to be more sensitive, which will be key to maximizing the return on a screening investment. New viral packaging systems may also enable size limitations for cDNAs in viral vectors to be overcome. In sum, expect cDNA overexpression to rival RNAi in popularity within a few years
With different strengths and weaknesses, knockdown and overexpression are, of course, highly complementary approaches. Rescue of RNAi knockdown by transfecting the corresponding cDNA is widely recognized as strong confirmation of the sequence specificity of the RNAi construct.
While there is no need to confirm the sequence specificity in cDNA overexpression, other errors and artifacts are possible and confirmation of results by an independent method (RNAi) is highly desirable. Thus, one might predict that if overexpression enhances a particular phenotype, RNAi knockdown will modulate the phenotype in the "opposite" manner.
Much has already been invested in the technology necessary to do genome-scale RNAi knockdown screens, including laboratory automation, high-content imaging systems, screening formats, reporter assays, and vectors. All of these tools are available for application to cDNA overexpression screens. Furthermore, programs for sharing gene content within institutions will become increasingly common, so that investigators will no longer need to choose one or the other approach, but can do both.
Dr. Paul Todd is product manager for genomics at OpenBiosystems, Inc.