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Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

ABSTRACT: Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven weeks of age. There was a hierarchy of FMRP expression: neocortex > hippocampus > cerebellum > brainstem. The expression of FMRP was highest and least variable in the neocortex, whereas it was most variable in the hippocampus. Male C57Bl/6J and FVB mice were selected to determine FMRP developmental differences in the brain at 3, 7, 10, and 14 weeks of age. We examined the four structures and found a developmental decline in FMRP expression with age, except for the brainstem where it remained stable. qFMRPm assay of blood had highest values in 3 week old animals and dropped by 2.5-fold with age. Sex differences were not significant. The results establish qFMRPm as a valuable tool due to its ease of methodology, cost effectiveness, and accuracy.

SUBMITTER: Adayev T

PROVIDER: S-EPMC8535242 | biostudies-literature | 2021 Sep

REPOSITORIES: biostudies-literature

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Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

Adayev Tatyana T LaFauci Giuseppe G Xu Weimin W Dobkin Carl C Kascsak Richard R Brown W Ted WT Goodman Jeffrey H JH

Genes 20210926 10

Fragile X syndrome results from the absence of the FMR1 gene product-Fragile X Mental Retardation Protein (FMRP). Fragile X animal research has lacked a reliable method to quantify FMRP. We report the development of an array of FMRP-specific monoclonal antibodies and their application for quantitative assessment of FMRP (qFMRPm) in mouse tissue. To characterize the assay, we determined the normal variability of FMRP expression in four brain structures of six different mouse strains at seven week ...[more]

PMID: 34680911

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Project description:In vitro genotoxicity dose-response data have been investigated for their utility in modeling and assessing potential differences in mutagenic responses between machine-generated whole smoke solutions (WSSs) from combusted cigarette tobacco products. Our previous study observed that potency ranking by benchmark dose (BMD) analysis was a useful modeling approach for quantitative assessment of differences between the mutagenicity of several structurally diverse chemical constituents of cigarette smoke. To follow-up on these observations, we used the mouse lymphoma assay (MLA) to evaluate the mutagenicity of WSSs prepared from two commercial cigarettes smoked under two different smoking machine regimens. L5178Y cells were exposed to ≥5 concentrations of each WSS for 4 hr ± S9 activation. S9 reduced the cytotoxicity and enhanced the mutagenicity of the WSSs. The resulting S9-mediated mutagenicity dose-responses were compared between test articles using BMD analysis, the lowest dose exceeding the Global Evaluation Factor, the no observed or lowest observed genotoxic effect level, and the mutagenic potency. The BMD10 , BMD50 , BMD100 , and BMD200 , indicating a 10%, 50%, 100%, or 200% increase in the background mutant frequency, respectively, were calculated using the PROAST software package. Overall, the quantitative approaches resulted in a similar rank order of mutagenic potency for the MLA tested WSSs, with potency increasing with the level of tar. The BMD approach using covariate analysis produced the most informative comparisons. Differences in potency were associated with the number of cigarettes smoked, the cigarette product smoked, and the smoking machine protocol used to prepare the sample. Under the conditions of this study, these results suggest that our hypothesis of modeling MLA data using the BMD approach to quantitatively discriminate between the mutagenic potential of WSSs from combustible cigarettes might be an useful method. Environ. Mol. Mutagen. 59:103-113, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

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Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

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Development of a Quantitative FMRP Assay for Mouse Tissue Applications.

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