Highlights d Genome-wide resource for genetic mosaic analysis with double markers in mice d Resource for dissection of cell-autonomous gene function of >96% of the mouse genome d Resource for genome-wide analysis of genomic imprinting phenotypes d MADM chromosomes reveal non-random mitotic sister chromatid segregation in vivo

Chronic myeloid leukemia (CML) is a stem cell (SC) neoplasm characterized by the BCR/ABL1 oncogene. Although the disease can be kept under control using BCR/ABL1 tyrosine kinase inhibitors (TKIs) in most cases, some patients relapse or have resistant disease, so there is a need to identify new therapeutic targets in this malignancy. Recent data suggest that leukemic SCs (LSCs) in CML display the stem-cell (SC)-mobilizing cell surface enzyme dipeptidyl-peptidase IV (DPPIV = CD26) in an aberrant manner. In the present study, we analyzed the effects of the DPPIV blocker vildagliptin as single agent or in combination with the BCR/ABL1 TKI imatinib or nilotinib on growth and survival of CML LSCs in vitro and on LSC engraftment in an in vivo xenotransplantation nonobese diabetic SCID-IL-2Rγ (NSG) mouse model. We found that nilotinib induces apoptosis in CML LSCs and inhibits their engraftment in NSG mice. In contrast, no substantial effects were seen with imatinib or vildagliptin. Nevertheless, vildagliptin was found to reduce the "mobilization" of CML LSCs from a stroma cell layer consisting of mouse fibroblasts in an in vitro co-culture model, suggesting reduced disease expansion. However, although vildagliptin and nilotinib produced cooperative effects in individual experiments, overall, no significant effects of coadministered vildagliptin over nilotinib or imatinib treatment alone were seen on the engraftment of CML cells in NSG mice. Gliptins may be interesting drugs in the context of CML and nilotinib therapy, but our preclinical studies did not reveal a major cooperative effect of the drug-combination vildagliptin + nilotinib on engraftment of CML cells in NSG mice.

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Mosaic Analysis with Double Markers (MADM) offers a unique approach to visualize and concomitantly manipulate genetically-defined cells in mice with single-cell resolution. MADM applications include the analysis of lineage; single-cell morphology and physiology; genomic imprinting phenotypes; and dissection of cell-autonomous gene functions in vivo in health and disease. Yet, MADM could only be applied to <25% of all mouse genes on select chromosomes thus far. To overcome this limitation, we generated transgenic mice with knocked-in MADM cassettes near the centromeres of all 19 autosomes and validated their use across organs. With this resource, >96% of the entire mouse genome can now be subjected to single-cell genetic mosaic analysis. Beyond proof-of-principle, we applied our MADM library to systematically trace sister chromatid segregation in distinct mitotic cell lineages. We found striking chromosome-specific biases in segregation patterns, reflecting a putative mechanism for the asymmetric segregation of genetic determinants in somatic stem cell division.

Intra-bone marrow transplantation (IBMT) has been adapted for mouse models to improve the seeding efficiency of transplanted hematopoietic stem and progenitor cells. Commonly used injection volumes for IBMT into the tibia differ between 10 and 40 μL even though considerable amounts of injected cells leak into the blood circulation immediately after injection. Injection of 3 μL trypan blue into the tibia of dead BALB/c mice showed staining in large vessels of hind limbs, even without supporting circulation. We therefore tested the effective capacity of the medullary cavity of dissected tibiae and femora of different mouse strains by bioluminescence imaging after injection of luciferase expressing cells. Cell leakage was already observed at 3 μL of injection volume and the measured emission rate increased significantly when 5 and 10 μL of volume with the same cell concentration were injected. Surprisingly, increasing injection volumes containing constant cell amounts resulted in comparable emission rates, suggesting a similar amount of leaked and absorbed cells independent of the injection volume. However, the absorption of a specific amount of injected cells could not be confirmed, as the ratio of leaked to absorbed cells was similar between IBMT that were performed with a constant injection volume containing either low or high cell amounts. In summary, for optimal cell transplantation via IBMT in mice we suggest to inject a high concentrated cell suspension with a maximum injection volume of 3 μL.

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