Hexadimethrine Bromide

To generate pseudo-typed lentiviral particles, shRNA-encoding pLK4 vectors were transfected into 293 T cells along with pCL-Eco and Pax2 packaging vectors and VSV-G envelope vector. At 18–24 h prior to transfection, 7.5 × 10⁶ cells were seeded into one 15 cm tissue culture dish in cDMEM and incubated overnight. At 1–2 h prior to transfection, the media was replaced with fresh cDMEM. Lentiviral DNA was introduced into 293 T cells using the calcium phosphate transfection method. Per transfection, plasmids were combined at a ratio of 6:4:3 (30 ug lentiviral plasmid containing the desired construct, 20 ug packaging vectors, and 15 ug envelope vector) with CaCl₂ at a final concentration of 125 mM and HEPES-buffered saline at a final concentration of 50 mM HEPES pH 7.05, 140 mM NaCl, and 1.5 mM Na₂HPO₄). DNA was allowed to precipitate for 30 min at room temperature, then added drop-wise to 293 T cells. After 12–18 h, the transfection media was replaced with fresh cDMEM media combined with unsupplemented DMEM at a ratio of 20/80 for a final FBS concentration of 2%. Viral supernatant was harvested at 24 h intervals over the following 3 days, then spun down and filtered through 0.45-um Durapore Millex (Millipore) filters. Virus was diluted in a solution of 10% (w/v) PEG-8000 and 300 mM NaCl, rotated overnight at 4 °C, and spun down at 3000 g for 1 h at 4 °C. The viral pellet was held overnight at 4 °C in 1× PBS at 1/100th the original volume of the viral supernatant and spun down to pellet serum protein and other particulates. Concentrated virus was stored at -80 °C or used immediately to transduction. HUT 78 T lymphocytes at 0.5 × 10⁶ cells/mL were allowed to grow for 2–3 days in the presence of concentrated virus and hexadimethrine bromide (Polybrene) transfection reagent (Sigma Aldrich) at a concentration of 8 μg/mL before removal of the virus and initiation of puromycin selection. Cells were allowed to expand in the presence of increasing puromycin concentration, from 0.25 μg/mL to a final selection concentration of 1 μg/mL. Whole cell lysates were probed for loss of GRB2 expression and expression of GRB2 mutants after 2–3 passages in puromycin.

CRISPR/Cas9 TET2-targeted HEL cells were generated using a 1-vector system: pLV-U6-gRNA/EF1a-puro-2A-Cas9-2A-GFP in lentiviral particles (Sigma-Aldrich) with the sgRNA sequence 5′GTTTGGTGCGGGAGCGAGC3′ targeting TET2 exon 6 (TET2 transcript ID ENST00000540549.5). lentiviral particles were incubated with HEL cells (at MOI of 2) in CM supplemented with 8 μg/mL hexadimethrine bromide and centrifuged at 800g for 30 minutes at 32°C. Transduced cells were selected by culture in CM supplemented with 2 μg/mL puromycin, then subsequently cloned by plating in soft agar (CM supplemented with 0.2% agarose). DNA was extracted from cell clones using a QIAamp DNA Micro Kit (QIAGEN), and TET2 mutation was confirmed by Sanger sequencing of exon 4 as described below (Supplemental Table 9). Control cell clones (with monoallelic TET2 mutation) were derived from parental HEL cells transduced with virus carrying an empty vector. All the clones used in experiments were generated independently, and there is some heterogeneity in expression profiles and phenotype. Moreover, it should be noted that several independent cell clones were generated for each TET2 genotype, and not every clone was used in every experiment.