In order to accurately study protein function in living cells, we want to ensure that the regulation and expression of genes we modify are artificially perturbed as little as possible. To this end, we are employing both Cas9/CRISPR-based genome modification techniques, and BACs (large genomic DNA constructs) as transgenes. With the Cas9 nuclease we can target specific protein tags or mutations into the genome of the cells being studied. Similarly, BAC transgenes usually deliver physiological expression at endogenous levels because they retain all cis-regulatory elements in the native configuration, and also allow for alternative splice isoforms, translational and miRNA controls, and alternative polyadenylation sites. Both of these methods provide a more accurate way of probing protein function than traditional cDNA-based studies, which often employ viral promotors that are accompanied by problems with deregulation and over-expression.
We have developed new recombineering procedures to modify BACs, in order to tag them with fluorescent markers, introduce targeted point mutations, or modify them to be RNAi-resistant. This allows us to rapidly construct wild-type and mutated RNAi-resistant transgenes, whose gene products we can follow in living mammalian cells under endogenous regulation as the only version expressed in the cell.
We have also collaborated to develop immunoprecipitation-mass spectrometry protocols incorporating the advantages of BAC transgenes to quantitatively identify differences in protein interaction partners and complex formation in cells containing wild-type versus mutant proteins.
Ongoing work in the lab continues to modify and integrate both Cas9- and BAC-based techniques to better assay protein function in live cells.
Bird et al., Nat Methods, 2012
Poser, Sarov et al., Nat Methods, 2008
Hubner, Bird et al., JCB, 2010
Counterselection recombineering strategy
Genome Editing and Bacterial Artificial Chromosome (BAC) transgenesis techniques