Department of Pathology, University of Virginia School of Medicine, Charlottesville, Virginia, USA.
University of Colorado Denver, Anschutz Medical Campus, Denver, Colorado, USA.
Transfusion. 2021 Oct;61(10):3017-3025. doi: 10.1111/trf.16628. Epub 2021 Sep 3.
Genetically modified mice are used widely to explore mechanisms in most biomedical fields-including transfusion. Concluding that a gene modification is responsible for a phenotypic change assumes no other differences between the gene-modified and wild-type mice besides the targetted gene.
To test the hypothesis that the N-terminus of Band3, which regulates metabolism, affects RBC storage biology, RBCs from mice with a modified N-terminus of Band3 were stored under simulated blood bank conditions. All strains of mice were generated with the same initial embryonic stem cells from 129 mice and each strain was backcrossed with C57BL/6 (B6) mice. Both 24-h recoveries post-transfusion and metabolomics were determined for stored RBCs. Genetic profiles of mice were assessed by a high-resolution SNP array.
RBCs from mice with a mutated Band3 N-terminus had increased lipid oxidation and worse 24-h recoveries, "demonstrating" that Band3 regulates oxidative injury during RBC storage. However, SNP analysis demonstrated variable inheritance of 129 genetic elements between strains. Controlled interbreeding experiments demonstrated that the changes in lipid oxidation and some of the decreased 24-hr recovery were caused by inheritance of a region of chromosome 1 of 129 origin, and not due to the modification of Band 3. SNP genotyping of a panel of commonly used commercially available KO mice showed considerable 129 contamination, despite wild-type B6 mice being listed as the correct control.
Thousands of articles published each year use gene-modified mice, yet genetic background issues are rarely considered. Assessment of such issues are not, but should become, routine norms of murine experimentation.
基因修饰小鼠被广泛用于探索包括输血在内的大多数生物医学领域的机制。假设除了目标基因之外，基因修饰小鼠和野生型小鼠之间没有其他差异，从而得出基因修饰是表型变化的原因。
为了验证 N 端的 Band3 调节代谢，影响 RBC 储存生物学的假设，用 N 端修饰的 Band3 的 RBC 从携带修饰的 N 端的 Band3 的小鼠中分离出来，在模拟血库条件下储存。所有小鼠品系均由来自 129 只小鼠的相同初始胚胎干细胞生成，每种品系均与 C57BL/6（B6）小鼠回交。对储存的 RBC 进行输血后 24 小时恢复和代谢组学的测定。通过高分辨率 SNP 阵列评估小鼠的遗传谱。
Band3 N 端突变的 RBC 发生脂质氧化增加和 24 小时恢复率降低，“证明”Band3 调节 RBC 储存过程中的氧化损伤。然而，SNP 分析表明，不同品系之间的 129 个遗传因子的遗传存在差异。控制性杂交实验表明，脂质氧化的变化和一些 24 小时恢复率的降低是由 129 起源的染色体 1 区域的遗传引起的，而不是由于 Band3 的修饰。对一组常用的 KO 小鼠的 SNP 基因分型显示，尽管列出的正确对照是野生型 B6 小鼠，但存在大量的 129 污染。
每年发表的数千篇文章都使用基因修饰小鼠，但遗传背景问题很少被考虑。评估这些问题不应该，但应该成为小鼠实验的常规规范。
