Overexpression of LARGE by means of genetic or pharmacological intervention could restore ligand binding and improve muscle strength in patients affected by dystroglycanopathies. However, prior to a therapeutic strategy based on the over-expression of LARGE being considered, an assessment of the safety of its long term overexpression and its efficacy with respect to the hyperglycosylation of a-DG needs to be established in vivo. To this end we report here the generation of four lines of LARGE overexpressing transgenic mice. We have characterised the effect of transgene expression on a-DG glycosylation in skeletal and cardiac muscle and brain, Niraparib tissues which are affected in dystroglycanopathy patients, as well as other tissues not involved in these disorders. We show that the overexpression of LARGE results in a robust hyperglycosylation of a-DG in skeletal and cardiac muscle without any observable deleterious morphological effect. Detailed analysis of the contractile properties of tibialis anterior muscles however showed a loss of force in response to eccentric exercise in older mice. This was not accompanied by any morphological changes suggesting a mild subclinical defect. a-DG was not hyperglycosylated in brain despite low levels of expression of the transgene, which suggests that higher levels of LARGE are necessary to achieve hyperglycosylation in a tissue, in which high levels of endogenous Large are present. In order to generate transgenic mice we cloned human LARGE into the pCAGGS expression vector which contains a human cytomegalovirus enhancer situated upstream of the chicken b-actin promoter and a rabbit b-globin 39 flanking MLN4924 sequence including a polyadenylation signal. Since antibodies to human LARGE are not routinely available, the LARGE cDNA derived from total human brain RNA was initially cloned into the pcDNA 3.1/V5-His expression vector. This directs the synthesis of a fusion protein with the V5 epitope at the C terminal end. The DNA sequence coding for this fusion product was subsequently subcloned into pCAGGS. The transgene expression vector harbouring the LARGE/V5 fusion sequence was digested to release a 4kb cassette for micro injection. Founders were identified by PCR from ear biopsies and were used to establish independent transgenic lines by breeding to wild-type F1 hybrid mice. Successful transmission of the transagene was identified by a further round of PCR screening. Expression of the transgene was confirmed by western blot analysis and immunocytochemistry using a V5 antibody.