A C57BL/6J Fancg-KO Mouse Model Generated by CRISPR/Cas9 Partially Captures the Human Phenotype

Abstract

Fanconi anemia (FA) develops due to a mutation in one of the FANC genes that are involved in the repair of interstrand crosslinks (ICLs). FANCG, a member of the FA core complex, is essential for ICL repair. Previous FANCG-deficient mouse models were generated with drug-based selection cassettes in mixed mice backgrounds, leading to a disparity in the interpretation of genotype-related phenotype. We created a Fancg-KO (KO) mouse model using CRISPR/Cas9 to exclude these confounders. The entire Fancg locus was targeted and maintained on the immunological well-characterized C57BL/6J background. The intercrossing of heterozygous mice resulted in sub-Mendelian numbers of homozygous mice, suggesting the loss of FANCG can be embryonically lethal. KO mice displayed infertility and hypogonadism, but no other developmental problems. Bone marrow analysis revealed a defect in various hematopoietic stem and progenitor subsets with a bias towards myelopoiesis. Cell lines derived from Fancg-KO mice were hypersensitive to the crosslinking agents cisplatin and Mitomycin C, and Fancg-KO mouse embryonic fibroblasts (MEFs) displayed increased γ-H2AX upon cisplatin treatment. The reconstitution of these MEFs with Fancg cDNA corrected for the ICL hypersensitivity. This project provides a new, genetically, and immunologically well-defined Fancg-KO mouse model for further in vivo and in vitro studies on FANCG and ICL repair.

Dual role of proliferating cell nuclear antigen monoubiquitination in facilitating Fanconi anemia-mediated interstrand crosslink repair

“…Due to lack of a functional anti-FANCG antibody, we utilized a flagged FANCG overexpression system ( 39 ). Potential alterations in the FANCG interactome were addressed by rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) ( 49 ) (Figure 4 A).…”

“…The generation of Pcna +/K164R(KR) and Fancg −/− mice has been described elsewhere ( 39 , 40 , 77 ). Timed matings of Pcna KR/+ Fancg +/− females were set up with corresponding males to obtain embryos.…”

“…To address these subjects, we intercrossed Fancg +/− mouse model ( 39 ) with the preestablished PCNA K164R/+ model ( 40 , 41 ). A homozygous combination of both alterations [double mutants (DMs)] was found to be embryonically lethal, indicating that the lack of both DDT and FA is incompatible with mammalian life.…”