Lnk domains and Y536 contribute differently to the proliferation of hematopoietic progenitors in vitro. (A) Total RNA was extracted from wild-type (+/+) or Lnk^−/− Lin⁻GFP⁺ progenitor cells transduced with either vector alone, WT or mutant forms of Lnk and subjected to RT-PCR with specific primers for Lnk (top panel) or HPRT (bottom panel) as control. (B) Wild-type or Lnk^−/− Lin⁻GFP⁺ transduced cells were assessed for their in vitro proliferative capacity in methylcellulose media containing IL-7 and SCF. Data represent the mean plus or minus SD (error bars) of number of colonies per 10³ Lin⁻GFP⁺ cells from triplicate samples from 3 independent assays using 3 to 5 mice of each genotype. Statistical significance was determined using the Student t test: ***P ≤ .01. (C) Typical appearance of GFP⁺ pre-B colonies from Lin⁻ transduced progenitor cells in response to IL-7 + SCF after 7 days of incubation in methylcellulose media. GFP⁺ colonies were acquired with a 40×/0.55 NA dry objective and a charge-coupled device (CCD) camera (MicroMax 13004, Princeton Instruments, Trenton, NJ). Images were processed with MetaMorph software (Molecular Devices, Downingtown, PA). Scale bar equals 20 μm.