Molecular Cloning and Expression of cDNA for Murine Galactocerebrosidase and Mutation Analysis of the Twitcher Mouse, a Model of Krabbe's Disease

Abstract

The cDNA for a murine galactocerebrosidase was isolated from a murine testis cDNA library on the basis of its homology with the cDNA for human galactocerebrosidase and a PCR method was used to clone the 5′ end. It has a 2,278‐nucleotide sequence including a 2,004‐nucleotide open reading frame, which encodes 668 amino acid residues. The identity between the human and murine amino acid sequences was very high, being calculated to be 84%. Sequencing of cDNA from liver of the twitcher mouse revealed a nonsense mutation at codon 339 (TGG → TGA). The most abundant mRNA of the murine galactocerebrosidase gave a 3.6‐kb band, which was not detected in twitcher mice. This suggests that the cDNA (2,278 bp) we characterized represents a minor species generated by an alternate poly(A) signal and that most of the mRNA has a much longer 3′‐untranslated region. Genome analysis revealed that this mutation was homozygous in the twitcher and heterozygous in the carrier but was not present in normal mice. The normal mouse cDNA but not the mutant cDNA of the galactocerebrosidase transfected into COS1 cells gave rise to an increase in enzymatic activity. We concluded that this mutation results in the deficiency of galactocerebrosidase in the twitcher mouse.

Intraventricular administration of recombinant adenovirus to neonatal twitcher mouse leads to clinicopathological improvements

“…The genotypes were determined around PND 5 to 7 by a PCR method using DNA extracted from mouse tail. 12 Forty-seven twitcher mice (twi/twi) were divided into four groups: 15 mice received AxCAGALC injection intraventricularly at PND 0 (PND 0-GALC group); 15 received AxCAGALC injection at PND 15 (PND 15-GALC group); three received AxCALacZ injection at PND 0 and 14 untreated twitcher mice were taken as controls. Six normal mice received AxCALacZ injection at PND 0 (PND 0-LacZ group) and three at PND 15 (PND 15-LacZ group).…”

“…9 However, the disadvantages of BMT such as shortage in donor supply and the side-effects largely limit the clinical application of this treatment. With the success in the cloning of GALC cDNA and the identification of gene mutation in twitcher, [10][11][12] gene therapy of the twitcher mouse became possible and challenging.…”

The Myelin Mutants as Models to Study Myelin Repair in the Leukodystrophies

“…The mouse lacks the GALC enzyme due to a nonsense mutation in the GALC gene [66]. Affected twi mice (on B6 background) develop a tremor at P10, fail to thrive, and develop rapidly progressive neurological dysfunction with paresis leading to hind limb paralysis after P30.…”

Mouse Forward Genetics in the Study of the Peripheral Nervous System and Human Peripheral Neuropathy

“…In addition to sharing histological features, such as loss of myelin in both the CNS and PNS and abnormal cell morphology, the twi mouse demonstrates the biochemical features of human Krabbe disease: loss of galactosylceramidase activity resulting in the accumulation of the substrate galactosylsphingosine (psychosine), believed to be toxic [77]. Unsurprisingly, the genetic defect in the twi mouse was identified as a nonsense mutation in the galactosylceramidase (Galc) gene [78], the same gene affected in human patients [79].…”