A simple method for early age phenotype confirmation using toe tissue from a mouse model of MPS IIIA

Paul J. Trim, Adeline A. Lau, John J. Hopwood, Marten F. Snel

Research output: Contribution to journalArticlepeer-review

20 Citations (Scopus)

Abstract

RATIONALE Determination of genotype can be difficult, especially during the early stages of developing an animal model, e.g. when PCR primers are not yet available. An increase or decrease in specific metabolites can be used as a surrogate marker for genotype; for instance, in homozygous MPS IIIA mice heparan sulphate (HS) is increased. METHODS A simple method was developed for extracting and depolymerising HS from mouse toe tissue using methanolysis under acidic conditions. The sample was lyophilised and resuspended in methanolic HCl. The reaction products are desulphated disaccharides and readily analysable by liquid chromatography/tandem mass spectrometry (LC/MS/MS) in positive ion multiple reaction monitoring mode. Measurements were normalised to a spiked deuterated HS internal standard and to endogenous chondroitin sulphate (CS). RESULTS HS was measured in toe tissue taken from 30 mice in three groups of 10 (normal controls, MPS IIIA homozygotes and heterozygotes). A significant difference was observed between the MPS IIIA homozygotes and the other two groups, making it possible to identify mice with the MPS IIIA genotype based on the measurement of HS. Normalisation to CS was shown to correct for sample variability and reaction efficiency. CONCLUSIONS Analysis of toe tissue provides a simple and rapid way of determining a storage phenotype at 5 to 7 days of age. Significantly, this method does not require any additional samples to be taken from animals, as it utilises tissue that is a by-product of toe clipping, a method that is routinely used to permanently identify mice.

Original languageEnglish
Pages (from-to)933-938
Number of pages6
JournalRapid Communications in Mass Spectrometry
Volume28
Issue number8
DOIs
Publication statusPublished - 30 Apr 2014
Externally publishedYes

Keywords

  • age phenotype confirmation
  • Determination of genotype
  • heparan sulphate

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