Functional analysis of CSF3R mutants in myeloid progenitor cell assays. In vitro colony growth of Csf3r-deficient murine hematopoietic progenitor cells expressing different CSF3R mutants. (A) Graphical representation of the different CSF3R constructs. Wild-type (wt), T595I (containing the extracellular mutation at amino acid position 595), d715 (containing the intracellular mutation Q716X, causing the introduction of a stop codon at amino acid position 716), and T595I/d715, containing both mutations as found in the SCN/AML patient. Ig indicates the Ig-like domain; CRH, cytokine receptor homology domain; FNIII, fibronectin type III repeats; TM, transmembrane domain; and cyto, cytoplasmic domain. Nomenclature has been adopted from Layton et al.42 (B) Colonies were grown in the presence of puromycin without growth factor (no GF) or with 100 ng/mL of human G-CSF. The induced colony growth is dependent on the transduction efficiency and the type of CSF3R construct. The transduction efficiency can be deduced from the number of GM-CSF–induced colonies under puromycin selection, because the CSF3R constructs confer puromycin resistance, but do not affect GM-CSF–induced colony growth. Therefore, by dividing the number of colonies by the number of GM-CSF–induced colonies, the transduction efficiency was corrected for.
