In vitro phenotypic correction of hematopoietic progenitors from Fanconi anemia group A knockout mice

Abstract

Fanconi anemia (FA) is a rare autosomal recessive disease, characterized by bone marrow failure and cancer predisposition. So far, 8 complementation groups have been identified, although mutations in FANCA account for the disease in the majority of FA patients. In this study we characterized the hematopoietic phenotype of a Fanca knockout mouse model and corrected the main phenotypic characteristics of the bone marrow (BM) progenitors using retroviral vectors. The hematopoiesis of these animals was characterized by a modest though significant thrombocytopenia, consistent with reduced numbers of BM megakaryocyte progenitors. As observed in other FA models, the hematopoietic progenitors from Fanca−/− mice were highly sensitive to mitomycin C (MMC). In addition, we observed for the first time in a FA mouse model a marked in vitro growth defect ofFanca−/−progenitors, either when total BM or when purified Lin−Sca-1+ cells were subjected to in vitro stimulation. Liquid cultures ofFanca−/−BM that were stimulated with stem cell factor plus interleukin-11 produced low numbers of granulocyte macrophage colony-forming units, contained a high proportion of apoptotic cells, and generated a decreased proportion of granulocyte versus macrophage cells, compared to normal BM cultures. Aiming to correct the phenotype of Fanca−/−progenitors, purified Lin−Sca-1+ cells were transduced with retroviral vectors encoding the enhanced green fluorescent protein (EGFP) gene and human FANCAgenes. Lin−Sca-1+ cells fromFanca−/−mice were transduced with an efficiency similar to that of samples from wild-type mice. More significantly, transductions with FANCA vectors corrected both the MMC hypersensitivity as well as the impaired ex vivo expansion ability that characterized the BM progenitors ofFanca−/−mice.

Assembling an orchestra: Fanconi anemia pathway of repair

“…They established that BRCA2, a factor that facilitates formation of RAD51-ssDNA nucleofilaments (162,163), is not only mutated in FANCD1 patient (48), but also interacts with monoubiquitinated FANCD2 to form nuclear foci (139,140). FANCD1 (BRCA2) appears to operate downstream of the FA core complex, but FANCD1/BRCA2 is more important for the repair of replication-blocking lesions relative to the FA core complex (118,161,(164)(165)(166)(167)(168). Although the FANCD1/BRCA2 ∆27/∆27 deficient mice do not recapitulate the bone marrow failure characteristic of FA, their bone marrow cells display more severe spontaneous and crosslinker-induced chromosomal aberrations than the FANCA -/mice (164,169).…”

Unraveling DNA Repair Processes In Vivo: Insights from Zebrafish Studies

“…Over the past several decades, studies employing mouse models, specifically those with loss-of-function approaches, have demonstrated the importance of DNA repair pathways in vertebrate development [7]. The targeted investigation of specific genes has further enhanced our understanding, revealing their distinct roles in essential biological processes such as neurogenesis, hematopoiesis, and germ cell development [8][9][10][11][12][13]. However, despite these advances, the majority of genes involved in DNA repair have not been thoroughly examined using the knockout (KO) mice system, primarily due to the early embryonic lethality [14][15][16][17][18][19].…”