Lentiviral Expression of ANKRD26 Variants

cDNA encoding full-length or truncated exon11+ ANKRD26 and a WAC-ANKRD26 fusion starting from exon 10 of ANKRD26 were cloned into the HMD lentiviral backbone. For production of lentiviruses, the appropriate viral packaging and genomic vectors were introduced into HEK 293T cells with calcium phosphate transfection. Viral supernatants were collected 48 h after transfection. To examine the activity of the fusion transcript-encoded cDNAs, overexpression in HSPCs was performed by transduction of relevant constructs at a multiplicity of infection of 50. HSPCs were cultured for 5 d after transduction in serum-free StemSpan II medium (STEMCELL Technologies) supplemented with CC100 cytokine cocktail (STEMCELL Technologies), 50 ng/ml TPO (PeproTech), and 35 nM UM171 (Bao et al., 2020 (link)). To assess for effects upon signaling activity downstream of the TPO receptor, 96 h after transduction cells were serum starved for 2 h followed by stimulation with TPO at 50 ng/ml for 15 min and subjected to downstream analysis.