3170 - Variation in HoxA9 Expression in the NUP98-HOXD13 Myelodysplasia Model, Using a Novel HoxA9-eGFP Reporter Mouse

Abstract

HoxA9 is normally expressed in haematopoietic stem and progenitor cells of humans and of mice, but expression ceases in differentiated cells. Many patients with a variety of myeloid and lymphoid malignancies overexpress HoxA9 in their disease cells, a fact reflected in many mouse models of these diseases. HoxA9 overexpression is thought to drive a differentiation block or abnormal self-renewal in these cells, promoting the oncogenic phenotype. We sought to produce a transgenic mouse that reports expression of the HoxA9 gene from the endogenous locus via co-expression of a fluorescent marker, in hopes that this might facilitate the better understanding of the origins of disease. We designed a targeting strategy using CRISPR-Cas9 technology, inserting an eGFP cassette immediately before the endogenous HoxA9 stop codon, separated from the HoxA9 coding sequence by a P2A self-cleaving sequence. We used this strategy to generate transgenic mice and found that the resultant mice contained the expected sequence and were developmentally normal and fertile. We characterised the expression of eGFP in the haematopoietic system of 6 week old mice using flow cytometry and found that, as expected, mature blood cells were eGFP negative while within the stem and progenitor compartments there were consistent, discrete HoxA9-positive and -negative compartments. We sorted these cells and confirmed by RQ-PCR analysis that eGFP expression did indeed correlate with expression of HoxA9. We subsequently crossed these mice with NUP98-HOXD13 transgenic mice, an established model of MDS characterised by HoxA9 overexpression in progenitor and mature cells, and found a markedly higher percentage of eGFP positive stem and progenitor cells, and that the positive cells were also markedly brighter. eGFP expression levels within the stem and progenitor compartments were more variable in the NHD13 mice, perhaps suggesting variable growth of a clonal population. As expected, we also observed expression of eGFP in certain mature cell compartments. These mice represent a valuable tool for studying the role of HoxA9 in haematopoiesis.