title: Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains.
abstract: 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.
authors.affiliation: Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
authors.forename: R
authors.initials: R
authors.lastname: Szabo
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia.
authors.forename: A L
authors.initials: AL
authors.lastname: Samson
authors.affiliation: Internal Medicine, Division of Cardiovascular Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Lawrence
authors.affiliation: Australian Centre for Blood Diseases, Monash University, Alfred Medical Research and Education Precinct, Melbourne, Victoria, Australia.
authors.forename: R L
authors.initials: RL
authors.lastname: Medcalf
authors.affiliation: Proteases and Tissue Remodeling Section, Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD, USA.
authors.forename: T H
authors.initials: TH
authors.lastname: Bugge
mesh_terms: D000483:Alleles; D000818:Animals; D001921:Brain / Q000378:metabolism; D002875:Chromosomes / Q000648:ultrastructure; D019295:Computational Biology; D003433:Crosses, Genetic; D053595:Embryonic Stem Cells / Q000166:cytology; D005091:Exons; D005260:Female; D005342:Fibrinolysis; D000072669:Gene Editing; D005786:Gene Expression Regulation; D018390:Gene Targeting; D005838:Genotype; D008297:Male; D051379:Mice; D020297:Mice, Congenic; D008810:Mice, Inbred C57BL; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D021382:Protein Sorting Signals; D057057:Serine Proteases / Q000378:metabolism; D010959:Tissue Plasminogen Activator / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D021382:Protein Sorting Signals; D057057:Serine Proteases; D010959:Tissue Plasminogen Activator
keywords: brain; congenic mice; gene targeting; mutation; tissue plasminogen activator