Extended Data Fig. 3: Generation of the isogenic cell lines carrying BAG3 variants and a 3xFLAG epitope tag fusion in the endogenous copy of the BAG3 gene.
From: Functional analysis of a common BAG3 allele associated with protection from heart failure
(a) Workflow for the cell line generation. (b) Strategy for the insertion of a 3xFLAG epitope fusion at the C-terminal of the BAG3 gene. The BAG3C151R-FLAG variant was generated using the same process on a preexisting cell line bearing the C151R mutation. To generate the BAG3E455K-FLAG cell line, the homology arms were engineered to contain the SNP and insert it during recombination. (C-D) Genotypes of the single-cell clones picked for 3xFLAG insertion (c) and the co-segregation of the BAG3E455K variant (d). (e) Genotyping the products of the 3xFLAG insertion by PCR. These genotyping reactions was performed at least twice with identical results. (f) Cells with a heterozygous insertion of the 3xFLAG epitope tag also had a SNP in the other allele that extended the BAG3 protein product by 4 amino acids. (G-H) A droplet digital PCR phasing test was used to select clones that contained the desired SNP variants and the 3xFLAG C-terminal sequence in the same allele. The test used different probes (g) to generate an estimate of linked molecules for each cell line and probe combination (H; See Methods for more details) (i) Insertion of the 3xFLAG fusion in the BAG3 gene did not alter the protein levels. N = 4 and N = 3 cell extracts from separate differentiation batches; analysis performed using a one-way ANOVA. Data is presented as mean values +/− SEM.
