Szabo R, Samson A L, Lawrence D A, Medcalf R L, Bugge T H
Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia.
J Thromb Haemost. 2016 Aug;14(8):1618-28. doi: 10.1111/jth.13338. Epub 2016 Jun 20.
Essentials C57BL/6J-tissue plasminogen activator (tPA)-deficient mice are widely used to study tPA function. Congenic C57BL/6J-tPA-deficient mice harbor large 129-derived chromosomal segments. The 129-derived chromosomal segments contain gene mutations that may confound data interpretation. Passenger mutation-free isogenic tPA-deficient mice were generated for study of tPA function.
Background The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A number of neurological abnormalities have been reported in tPA-deficient mice. Objectives To study genetic contamination of tPA-deficient mice. Materials and methods Whole genome expression array analysis, RNAseq expression profiling, low- and high-density single nucleotide polymorphism (SNP) analysis, bioinformatics and genome editing were used to analyze gene expression in tPA-deficient mouse brains. Results and conclusions Genes differentially expressed in the brain of Plat(-/-) mice from two independent colonies highly backcrossed onto the C57BL/6J strain clustered near Plat on chromosome 8. SNP analysis attributed this anomaly to about 20 Mbp of DNA flanking Plat being of 129 origin in both strains. Bioinformatic analysis of these 129-derived chromosomal segments identified a significant number of mutations in genes co-segregating with the targeted Plat allele, including several potential null mutations. Using zinc finger nuclease technology, we generated novel 'passenger mutation'-free isogenic C57BL/6J-Plat(-/-) and FVB/NJ-Plat(-/-) mouse strains by introducing an 11 bp deletion into the exon encoding the signal peptide. These novel mouse strains will be a useful community resource for further exploration of tPA function in physiological and pathological processes.
要点 C57BL/6J-组织型纤溶酶原激活剂（tPA）缺陷小鼠被广泛用于研究tPA功能。同基因C57BL/6J-tPA缺陷小鼠携带源自129品系的大片段染色体。源自129品系的染色体片段包含可能混淆数据解读的基因突变。为了研究tPA功能，构建了无乘客突变的同基因tPA缺陷小鼠。
背景 在小鼠中产生明确的无效突变的能力彻底改变了哺乳动物基因功能的分析。然而，使用源自129品系的胚胎干细胞产生的基因缺陷小鼠可能携带大片段的129 DNA，即使与参考品系如C57BL/6J进行广泛回交也是如此，这可能会混淆在这些小鼠中进行的实验的解读。由PLAT基因编码的组织型纤溶酶原激活剂（tPA）是一种在大脑中广泛表达的纤维蛋白溶解丝氨酸蛋白酶。在tPA缺陷小鼠中已经报道了许多神经学异常。目的 研究tPA缺陷小鼠的基因污染。材料和方法 使用全基因组表达阵列分析、RNA测序表达谱分析、低密度和高密度单核苷酸多态性（SNP）分析、生物信息学和基因组编辑来分析tPA缺陷小鼠大脑中的基因表达。结果和结论 来自两个独立群体且高度回交至C57BL/6J品系的Plat(-/-)小鼠大脑中差异表达的基因聚集在8号染色体上靠近Plat的位置。SNP分析将这种异常归因于两个品系中Plat侧翼约20 Mbp的DNA源自129品系。对这些源自129品系的染色体片段进行生物信息学分析，在与靶向的Plat等位基因共分离的基因中鉴定出大量突变，包括几个潜在的无效突变。使用锌指核酸酶技术，通过在编码信号肽的外显子中引入11 bp缺失，构建了新的无“乘客突变”的同基因C57BL/6J-Plat(-/-)和FVB/NJ-Plat(-/-)小鼠品系。这些新的小鼠品系将成为进一步探索tPA在生理和病理过程中功能的有用的公共资源。
