Relevant Antibodies

 

FKRP antibodies

Polyclonal rabbit anti-FKRP antibodies were described by Derek Blake’s and Ichizo Nishino’s labs (Esapa et al., 2002 and Matsumoto et al, 2004). Also, Santa Cruz Biotechnology commercializes a goat-polyclonal anti-FKRP that detects recombinant FKRP from mouse, rat and human. However, these antibodies do not seem able to detect endogenous FKRP protein.

 

AbCam, Sigma and Novus Biologicals sell a rabbit polyclonal against human FKRP (predicted to react with mouse and rat) supposed to react with the endogenous protein, but so far there have been no publications citing its use.

 

An antibody against zebrafish FKRP was recently described by the Kunkel lab (Kawahara et al, 2010).

 

Alpha-dystroglycan antibodies

There are two antibodies that recognize glycosylated alpha-dystroglycan (IIH6 and VIA4 monoclonal antibodies), both developed in Kevin Campbell’s lab. Both antibodies are commercialized by Millipore (however, there are reports of significant batch to batch variation with these antibodies).

 

The Campbell lab has also developed a rabbit polyclonal antibody (GT-20) against the core of alpha-dystroglycan protein.

 

Animal Models

 

Targeted deletion of the mouse FKRP gene leads to embryonic lethality, but two labs have published mouse models of FKRP mutations. While mouse (knock-in) models based on patient FKRP mutations that result in MEB (Y307N), WWS (E310del) and MDC1C (P448L) have been generated, only those retaining the neomycin cassette succeeded in reducing Fkrp transcript levels and recapitulating the severity of the disease phenotypes.

Of the three mouse models, the E310del was embryonically lethal, FKRP-NeoTyr307Asn resulted in perinatal lethality, while some FKRP-neo-P448L mutant mice survived to 10 months, showing severe muscle pathology and CNS abnormalities (Chan et al, 2010; Ackroyd et al 2009). The difference in lifespan of these models could be associated with the level of Fkrp mRNA/protein present. Homozygous E310del mice completely lack functional FKRP due to deletion of the C-terminus of FKRP that contains a consensus DxD motif commonly found in glycosyltransferases, suggesting that the presence of at least some FKRP activity is critical for embryonic development. In contrast, the FKRP-NeoTyr307Asn homozygotes expressed ~40% of mutated Fkrp mRNA compared to controls, while FKRP-neo-P448L homozygotes expressed ~55%. This is consistent with results from knockdown of Fkrp transcript using RNA interference, which showed that the greater reductions of Fkrp mRNA (up to 75%) using dual shRNA cassettes induces overt dystrophic pathology, compared with the lower reductions from using single shRNA cassettes (Wang et al, 2011).

 

Susan Brown’s lab has recently generated a novel (unpublished) model that presents a muscle phenotype, but does not show central nervous involvement (which was the cause for their perinatal death), by crossing the Y307N mice with a Sox-1-Cre mouse (the neo cassette is floxed in the CNS so their brains develop normally). This new model, called FKRP MD, presents a near normal lifespan and shows a marked muscle phenotype by 12 weeks of age.

 

In Qi Lu’s lab, a new line was derived from the FKRP-neo-P448L by deleting the neo cassette. Homozygous FKRP-P448L mice present a less severe phenotype than those with the Neo cassette, have a near-normal life-span and can breed, contrary to FKRP-neo-P448L mice. These mice seem to reproduce disease features, such as skeletal muscle wasting and cardiac problems, but a more detailed characterization still remains to be done.

 

In addition, several labs have generated a knockin mouse model carrying the L276I missense mutation commonly seen in LGMD2I patients. These mice present a near normal life span and a very mild disease phenotype.  In order to create a more relevant pre-clinical model, this model is now being crossed with the E310del mouse in Qi Lu’s lab, in order to generate compound heterozygous mouse carrying one L276I allele and an E310del allele. These mice seem to have an intermediate phenotype, presenting both skeletal muscle and cardiac disease. However, this model has yet to be thoroughly characterized.