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At Cellecta, we have relied primarily on lentiviral vectors to develop our RNAi genetic screening technology. The broad tropism of lentivirus-based plasmids packaged into VSV-g pseudotyped viral particles provides a very convenient vector system to introduce and stably express shRNA expression constructs or constructs expressing small proteins, peptides, or other biological molecules into virtually any mammalian cell line. Unlike with most retroviruses, cells do not need to be dividing take up a lentivirus, and unlike adenoviruses, the lentiviral expression construct integrates into the cell's genomic DNA so it is stably passed onto progeny allowing long term studies over several cell passages. Also, in contrast with advenoviral vectors, lentiviral plasmids can be easily engineered allowing construction of complex pooled libraries such as those we use for RNAi screening.
However, there are some cells for which lentiviral vectors seem to work poorly. In particular, mouse hematopoietic cells appear to sometimes shut down expression of lentiviral constructs. We have found this anecdotally based on feedback from researchers using our system, and it has been noted in published literature. To address this, we looked into developing a more effective vector for stable shRNA expression in these types of cells.
We adapted a murine stem cell viral (MSCV) plasmid with our shRNA cloning cassette, which incorporates the U6 promoter to drive hairpin expression. As with our standard lentiviral constructs, the MSCV shRNA vector constitutively expresses a fluorescent protein marker (tagRFP) and puromycin antibiotic selection gene on a single transcript separated by a 2A self-cleaving peptide. These features enable easy assessment of infection rates and selection for transduced shRNA-expressing cells.
To test knockdown, we cloned an shRNA known to effectively knock down mouse p53 into the MSCV plasmid. Vikram Paralkar in Mitch Weiss's lab at the Children's Hospital of Philadelphia tested the effectiveness of the construct in erythroid progenitor cells extracted from day 14.5 CD-1 mouse embryo fetal livers. The erythroid progenitors were infected with the MSCV-p53 construct, as well a control construct that did not contain the p53 sequence, packaged in viral particles. A multiplicity of infection (MOI) of virus to cells was used such that approximately 90% of the cells were transduced. Then, after 48 hours, the cells were harvested and RT-PCR was used to assess the expression level of the p53 target gene. The results below show that at least 75% knockdown of the p53 target transcript was obtained.
The results indicate that this MSCV vector provides an effective alternative for shRNA expression in mouse hematopoietic cells where lentivirus infection is problematic. The efficiency levels of cloning and transduction with the MSCV vector appear to be sufficient to enable construction and screening with complex pooled shRNA libraries as effectively as with our standard lentiviral system.
