Evaluation of Cre Recombinase Models for the Study of Dnmt3a Mutations and Myeloid Bias in Clonal Hematopoiesis

Abstract

The process of aging has been found to have lasting effects on hematopoietic stem cells and their ability to maintain the homeostasis of the hematopoietic system. From the accumulation of DNA damage to the biased differentiation potential toward myeloid lineages, the effects of HSC aging have been shown to disrupt normal HSC function and result in hematological malignancies and illness. Additional research on the repercussions of HSC aging must be conducted to fully understand its association with hematological disease and the mechanisms underlying this disease development. The current paper evaluates the efficacy of Cre recombinase models developed to examine the age-related myeloid bias and the frequent mutation of Dnmt3a associated with aged HSCs.

The first project investigated the ability of a Cre recombinase system to generate Dnmt3a R878H mutations in HSCs through the excision of the endogenous R878H exon 23 and the subsequent expression of fluorescent protein tdTomato. Due to a size difference in the respective regions controlling the expression of tdTomato and the wildtype R878H exon 23, exposure to Cre recombinase could result in the isolated expression of tdTomato without the coinciding expression of mutant Dnmt3a. Using FACSfacilitated single-cell isolation and expansion, HSCs were collected from TAM-treated experimental mice and genetically tested for the expression of both tdTomato and the Dnmt3a R878H mutation with PCR. Although Cre recombinase exposure was expected to result in the expression of the mutant Dnmt3a, primers detecting Dnmt3a wild-type and mutant exons were only able to generate bands corresponding to the wild-type exon 23 for the isolated HSC DNA. Similarly, primers specifically detecting the excision of the Dnmt3a wild-type were also unable to produce any bands. Overall, despite efforts to troubleshoot and optimize PCR performance, western blots examining the excision of the endogenous Dnmt3a exon were unable to generate sufficient results, suggesting an issue with the DNA isolation protocol or with the Dnmt3a excision primers themselves.

The second project examined a Cre recombinase system designed to simulate the myeloid dominance observed in older individuals through the depletion of multipotent progenitors. Embryonic multipotent progenitors have recently been identified as a significant but gradually decreasing source of lymphoid cells, implicating the cell type in the aged body’s reduced presence of lymphoid cells and characteristic myeloid skew. Peripheral blood samples from experimental and control mice were examined using flow cytometry for potential changes to the myeloid and lymphoid cell types over a threemonth period. In experimental samples, the CD4 T cell population was observed increasing upon the initial depletion of MPPs. However, this growth eventually resolved and experimental T cell counts lowered to those seen in controls. Similar results were also observed in the production of CD8 T cells. Additionally, experimental mice had generally high, unchanging CD19 B cell levels. All in all, more research is needed to further understand the changes observed in this project and the repercussions of MPP depletion on myeloid and lymphoid cells.