ABSTRACT
Proteins of interest are frequently expressed with a fusion-tag to facilitate experimental analysis. In trypanosomatids, which are typically diploid, a tag-encoding DNA fragment is typically fused to one native allele. However, since recombinant cells represent âª0.1% of the population following transfection, these DNA fragments also incorporate a marker cassette for positive selection. Consequently, native mRNA untranslated regions (UTRs) are replaced, potentially perturbing gene expression; in trypanosomatids, UTRs often impact gene expression in the context of widespread and constitutive polycistronic transcription. We sought to develop a tagging strategy that preserves native UTRs in bloodstream-form African trypanosomes, and here we describe a CRISPR/Cas9-based knock-in approach to drive precise and marker-free tagging of essential genes. Using simple tag-encoding amplicons, we tagged four proteins: a histone acetyltransferase, HAT2; a histone deacetylase, HDAC3; a cleavage and polyadenylation specificity factor, CPSF3; and a variant surface glycoprotein exclusion factor, VEX2. The approach maintained the native UTRs and yielded clonal strains expressing functional recombinant proteins, typically with both alleles tagged. We demonstrate utility for both immunofluorescence-based localisation and for enriching protein complexes; GFPHAT2 or GFPHDAC3 complexes in this case. This precision tagging approach facilitates the assembly of strains expressing essential recombinant genes with their native UTRs preserved.
Subject(s)
Trypanosoma brucei brucei , Trypanosoma , CRISPR-Cas Systems , Genes, Essential , Trypanosoma/genetics , Trypanosoma brucei brucei/genetics , Untranslated RegionsABSTRACT
BACKGROUND: Some PRKAG2 mutations in the human gene encoding for the gamma-subunit of the adenosine monophosphate-activated protein kinase (AMPK) recently have been shown to cause rhythm disturbances (often fatal) in affected patients. METHODS AND RESULTS: Rat ventricular myocytes were infected with an adenoviral vector designed to express a truncated constitutively active mutant (T172D) of the AMPK alpha1-subunit (CA-AMPK). The human cardiac sodium channel hH1 and CA-AMPK were also coexpressed in a mammalian cell line. Patch-clamp techniques were used to measure myocyte action potentials and recombinant hH1 sodium channel currents. Our results demonstrate that action potential duration is significantly prolonged in myocytes expressing the CA-AMPK construct, leading to the production of potentially arrhythmogenic early afterdepolarizations. Recombinant sodium channel current analysis revealed that expression of CA-AMPK significantly slowed open-state inactivation and shifted the voltage-activation curve in a hyperpolarizing direction. CONCLUSIONS: We propose that sodium channels may be substrates for AMPK, possibly contributing to the observed arrhythmogenic activity in patients with some PRKAG2 mutations.