Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

Daniel J. Rawle; Thuy T. Le; Troy Dumenil; Cameron Bishop; Kexin Yan; Eri Nakayama; Phillip I. Bird; Andreas Suhrbier

doi:10.7554/ELIFE.70207

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

Daniel J. Rawle, Thuy T. Le, Troy Dumenil, Cameron Bishop, Kexin Yan, Eri Nakayama, Phillip I. Bird, Andreas Suhrbier

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11 Citations (Scopus)

Abstract

Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma^-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma^-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is trun-cated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma^-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J Gzma^S211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteo-lytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

Original language	English
Article number	e70207
Number of pages	31
Journal	eLife
Volume	11
DOIs	https://doi.org/10.7554/ELIFE.70207
Publication status	Published - Feb 2022

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10.7554/ELIFE.70207Licence: CC BY

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@article{7abbfa8dc3d64241b42204888dfb7ed2,

title = "Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies",

abstract = "Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA{\textquoteright}s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is trun-cated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA{\textquoteright}s bioactivity free of background issues, with circulating proteo-lytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.",

author = "Rawle, {Daniel J.} and Le, {Thuy T.} and Troy Dumenil and Cameron Bishop and Kexin Yan and Eri Nakayama and Bird, {Phillip I.} and Andreas Suhrbier",

note = "Funding Information: We thank the following staff at QIMR Berghofer MRI for their assistance; animal house staff, Dr I Anraku (BSL3 facility manager), Dr R Johnston (Bioinformatics), and Dr Viviana Lutzky (for proof reading). We thank Dr Dion Kaiserman (Monash University, Australia) for supplying recombinant GZMA. We also thank Dr Mark Heise (University of North Carolina) for valuable discussions. National Health and Medical Research Council- APP1141421- Andreas Suhrbier; National Health and Medical Research Council- APP1173880- Andreas Suhrbier. Publisher Copyright: ‍{\textcopyright} Rawle et al.",

year = "2022",

month = feb,

doi = "10.7554/ELIFE.70207",

language = "English",

volume = "11",

journal = "eLife",

issn = "2050-084X",

publisher = "eLife Sciences Organisation, Ltd.",

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TY - JOUR

T1 - Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

AU - Rawle, Daniel J.

AU - Le, Thuy T.

AU - Dumenil, Troy

AU - Bishop, Cameron

AU - Yan, Kexin

AU - Nakayama, Eri

AU - Bird, Phillip I.

AU - Suhrbier, Andreas

N1 - Funding Information: We thank the following staff at QIMR Berghofer MRI for their assistance; animal house staff, Dr I Anraku (BSL3 facility manager), Dr R Johnston (Bioinformatics), and Dr Viviana Lutzky (for proof reading). We thank Dr Dion Kaiserman (Monash University, Australia) for supplying recombinant GZMA. We also thank Dr Mark Heise (University of North Carolina) for valuable discussions. National Health and Medical Research Council- APP1141421- Andreas Suhrbier; National Health and Medical Research Council- APP1173880- Andreas Suhrbier. Publisher Copyright: ‍© Rawle et al.

PY - 2022/2

Y1 - 2022/2

N2 - Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is trun-cated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteo-lytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

AB - Granzyme A (GZMA) is a serine protease secreted by cytotoxic lymphocytes, with Gzma-/- mouse studies having informed our understanding of GZMA’s physiological function. We show herein that Gzma-/- mice have a mixed C57BL/6J and C57BL/6N genetic background and retain the full-length nicotinamide nucleotide transhydrogenase (Nnt) gene, whereas Nnt is trun-cated in C57BL/6J mice. Chikungunya viral arthritis was substantially ameliorated in Gzma-/- mice; however, the presence of Nnt and the C57BL/6N background, rather than loss of GZMA expression, was responsible for this phenotype. A new CRISPR active site mutant C57BL/6J GzmaS211A mouse provided the first insights into GZMA’s bioactivity free of background issues, with circulating proteo-lytically active GZMA promoting immune-stimulating and pro-inflammatory signatures. Remarkably, k-mer mining of the Sequence Read Archive illustrated that ≈27% of Run Accessions and ≈38% of BioProjects listing C57BL/6J as the mouse strain had Nnt sequencing reads inconsistent with a C57BL/6J genetic background. Nnt and C57BL/6N background issues have clearly complicated our understanding of GZMA and may similarly have influenced studies across a broad range of fields.

UR - http://www.scopus.com/inward/record.url?scp=85124174169&partnerID=8YFLogxK

U2 - 10.7554/ELIFE.70207

DO - 10.7554/ELIFE.70207

M3 - Article

C2 - 35119362

AN - SCOPUS:85124174169

SN - 2050-084X

VL - 11

JO - eLife

JF - eLife

M1 - e70207

ER -

Widespread discrepancy in Nnt genotypes and genetic backgrounds complicates granzyme A and other knockout mouse studies

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Chikungunya virus disease; the role of proteases and their receptors

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