Secondary transplantation of SCL-deleted HSCs. (A) Comparison of donor contribution to myeloid, B, and T cells in primary (1) and secondary (2) transplants. Donor cells for primary transplantation were obtained from SCL^+/+ (wt, ▪), MxSCL^+/loxP (het, ▦), or MxSCL^-/loxP (deleted, □) mice 6 days after poly(I:C) administration. Secondary recipients received pooled bone marrow cells from primary recipients 4 months after transplantation. The mean and SD of 3 to 5 recipients are shown. The dashed line indicates the expected donor contribution (80%). (B) PCR genotype analysis of donor-derived myeloid or B cells in secondary recipients of donor bone marrow cells from poly(I:C)-treated MxSCL^+/loxP (het) or MxSCL^-/loxP (del) mice. FACS-isolated cells were used for genotyping. The PCR was designed to amplify only the loxP-targeted (loxP) and deleted loxP (Δ) SCL alleles. The control sample was a 50:50 mix of loxP and Δ alleles. (C) Southern blot of bone marrow (B), spleen (S), and thymus (T) from 2 secondary recipients 4 months after transplantation with marrow from primary recipients reconstituted with SCL-deleted donor cells and SCL-wild-type competitor cells. Genomic DNA was probed with a genomic fragment that can distinguish wild-type (+), loxP-targeted (loxP), deleted loxP (Δ), and null (-) SCL alleles. Note the skewing of SCL-deleted hematopoiesis, especially in recipient no. 2 where bone marrow cells are predominantly competitor (+) while the thymus is completely SCL-deleted (-/Δ).