Competitive Engraftment Assay of Camkk2 KSL Cells

For competitive engraftment assays, the total BM cells were isolated from femurs and tibiae of WT and Camkk2 null (CD45.2 background). c-Kit-positive cells were enriched using anti-CD117/c-Kit microbeads (Miltenyi Biotec, Auburn, CA, USA). The KSL CD34^- stem cells (CD45.2) were then sorted and 1000 cells were injected with 5x10⁵ competitor whole BM (CD45.1) into lethally irradiated (split dose totaling 10Gy) recipient B6-CD45.1 (B6.SJL-Ptprca Pepcb/BoyJ) mice via the retro-orbital sinus. In the same experiments, KSL CD34^- cells were sorted from BM of Camkk2 null or control mice treated with 200cGy 90 days before transplant. For lineage analysis, peripheral blood cells were collected and prepared as described.^{51 (link)} In some experiments, transplanted mice were monitored for up to 4 months, and then mice received 450cGy TBI. Mice were finally monitored for chimerism.