A MURINE AP-ENDONUCLEASE GENE-TARGETED DEFICIENCY WITH POSTIMPLANTATION EMBRYONIC PROGRESSION AND IONIZING-RADIATION SENSITIVITY

Apurinic/apyrimidinic endonuclease there designated APE/REF) carries o ut repair incision at abasic or single-strand break damages in mammals . This multifunctional protein also has putative role(s) as a cysteine 'reducing factor' (REF) in cell-stress transcriptional responses. To assess the significance of APE/REF for embryonic teratogenesis we cons tructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/R EF protein synthesis while retaining the Ape/Ref bi-directional promot er that avoided potential inactivation of an upstream gene. Chimeric a nimals crossed into Tac:N:NIHS-BC produced germline transmission. Homo zygous null Ape/Ref-embryos exhibited successful implantation and near ly normal developmental progression until embryonic day 7.5 followed b y morphogenetic failure and adsorption of embryos by day 9.5. We chara cterized the cellular events proceeding to embryonic lethality and exa mined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of puta tive Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo prope r-the epiblast. Increased cell-nucleus degeneration occurred within ep iblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ine ffective for replacement of lost cells. Extra-embryonic yolk sac, a tr ophectoderm derived lineage retained normal appearance to day 9, Expla nted homozygous Ape/Ref-null blastocysts displayed increased sensitivi ty to gamma-irradiation, most likely a manifestation of APE/REF incisi on defect. Our study establishes that this new Ape/Ref deficiency geno type is definitely capable of post-implantation developmental progress ion to the onset of gastrulation. Function(s) of APE/REF in base damag e incision and also conceivably in mitogenic responses towards epiblas t cell death are critical for transit through the gastrulation stage o f embryonic growth and development. (C) 1998 Elsevier Science B.V. All rights reserved.