Ludwig D L, MacInnes M A, Takiguchi Y, Purtymun P E, Henrie M, Flannery M, Meneses J, Pedersen R A, Chen D J
Life Sciences Division, Los Alamos National Laboratory, NM 87545, USA.
Mutat Res. 1998 Oct 21;409(1):17-29. doi: 10.1016/s0921-8777(98)00039-1.
Apurinic/apyrimidinic endonuclease (here designated APE/REF) carries out 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 constructed a more precisely targeted Ape/Ref-deficient genotype in mice. Ape/Ref gene replacement in ES cells eliminated the potential of APE/REF protein synthesis while retaining the Ape/Ref bi-directional promoter that avoided potential inactivation of an upstream gene. Chimeric animals crossed into Tac:N:NIHS-BC produced germline transmission. Homozygous null Ape/Ref-embryos exhibited successful implantation and nearly normal developmental progression until embryonic day 7.5 followed by morphogenetic failure and adsorption of embryos by day 9.5. We characterized the cellular events proceeding to embryonic lethality and examined ionizing radiation sensitivity of pre-implantation Ape/Ref-null embryos. After intermating of heterozygotes, Mendelian numbers of putative Ape/Ref-null progeny embryos at day 6.5 displayed a several-fold elevation of pycnotic, fragmenting cell nuclei within the embryo proper-the epiblast. Increased cell-nucleus degeneration occurred within epiblast cells while mitosis continued and before obvious morphogenetic disruption. Mitogenic response to epiblast cell death, if any, was ineffective for replacement of lost cells. Extra-embryonic yolk sac, a trophectoderm derived lineage retained normal appearance to day 9. Explanted homozygous Ape/Ref-null blastocysts displayed increased sensitivity to gamma-irradiation, most likely a manifestation of APE/REF incision defect. Our study establishes that this new Ape/Ref deficiency genotype is definitely capable of post-implantation developmental progression to the onset of gastrulation. Function(s) of APE/REF in base damage incision and also conceivably in mitogenic responses towards epiblast cell death are critical for transit through the gastrulation stage of embryonic growth and development.
脱嘌呤/脱嘧啶内切核酸酶（此处称为APE/REF）在哺乳动物中对无碱基或单链断裂损伤进行修复切割。这种多功能蛋白在细胞应激转录反应中还可能作为半胱氨酸“还原因子”（REF）发挥作用。为了评估APE/REF对胚胎致畸作用的重要性，我们构建了一种在小鼠中靶向性更精确的Ape/Ref基因缺失基因型。ES细胞中的Ape/Ref基因替换消除了APE/REF蛋白合成的可能性，同时保留了Ape/Ref双向启动子，避免了上游基因的潜在失活。嵌合体动物与Tac:N:NIHS-BC杂交产生了种系传递。纯合缺失的Ape/Ref胚胎在胚胎植入前表现出成功植入且发育进程几乎正常，直至胚胎第7.5天，随后在第9.5天出现形态发生失败和胚胎吸收。我们对导致胚胎致死的细胞事件进行了表征，并检查了植入前Ape/Ref缺失胚胎的电离辐射敏感性。杂合子杂交后，第6.5天推测为Ape/Ref缺失的后代胚胎数量显示，胚胎本体（即上胚层）内固缩、碎片化的细胞核数量增加了几倍。在上胚层细胞中，细胞核变性增加，而有丝分裂仍在继续，且在明显的形态发生破坏之前。如果存在的话，对上胚层细胞死亡的促有丝分裂反应对于替代丢失的细胞是无效的。胚胎外卵黄囊，一种滋养外胚层衍生的谱系，到第9天仍保持正常外观。移植的纯合Ape/Ref缺失囊胚对γ射线照射表现出更高的敏感性，这很可能是APE/REF切割缺陷的一种表现。我们的研究表明，这种新的Ape/Ref缺失基因型确实能够在植入后发育至原肠胚形成开始阶段。APE/REF在碱基损伤切割中的功能，以及可能在对上胚层细胞死亡的促有丝分裂反应中的功能，对于胚胎生长发育通过原肠胚形成阶段至关重要。
