BLOCK-iT Pol II miR RNAi system: Issues with generating stable knockdown - (Oct/13/2014 )
Hello,
For the last 6 months I, and others in my team, have been having a lot of difficulty generating stable transfectants using the BLOCK-iT Pol II miR RNAi Expression vector kit.
We prepared the miRNAs against our target gene transcript following the manufacturers instructions and cloned them into the pcDNATM6.2-GW/EmGFP miR vector that came with the kit. The constructs have been validated by restriction digest and DNA sequencing.
On multiple occasions we have transfected bovine embryonic fibroblasts (BEFs) with our miRNA constructs (including the positive and negative miRNA control plasmids supplied by the kit) using the lipofectamine LTX reagent as well as Neon Electroporation protocols. We typically see transient transfection efficiencies of 20 - 70% based on GFP expression. 48 hours after transfection we split the cells harshly (200,000 cells per 10cm plate) and start blasticidin selection the following day (10ug/ml) which we refresh every 2 days.
In the negative control dishes, which do not contain the miRNA construct, the cells all die during selection. However, in the miRNA transfected dishes (including the control plasmids supplied by the kit) we do not see much cell death and cells continue to grow to confluence. The majority of these cells are not GFP positive and cells which are GFP positive have not proliferated (i.e. no GFP cells adjacent to them).
It seems blasticidin resistance is occurring following transfection with the miRNA constructs but this does not correlate with GFP/miRNA expression. Yet, the BEF cells alone are not blasticidin resistant as the negative control cells all die within a few days of selection.
It would be nice to know if other people have experienced similar problems as there is nothing regarding this phenomenon in the troubleshooting section of the handbook. Why won't the few GFP cells proliferate after transfection??? and why are the non-GFP cells from transfected dishes blasticidin resistant????
As the non-transfected cells are dying, it seems that the system is working. Is it possible that the miRNA is affecting cell replication or major cellular pathways at all?... this could lead to your cells not proliferating.
Could your miRNA be targeting GFP? (probably not, but you never know until you test it) Could the plasmid be being methylated after integration? Are you sure the plasmid is integrating? Is there any chance that a recombination event is happening?