A dystrophic Duchenne mouse model for testing human antisense oligonucleotides.

Duchenne muscular dystrophy (DMD) is a severe muscle-wasting disease generally caused by reading frame disrupting mutations in the DMD gene resulting in loss of functional dystrophin protein. The reading frame can be restored by antisense oligonucleotide (AON)-mediated exon skipping, allowing production of internally deleted, but partially functional dystrophin proteins as found in the less severe Becker muscular dystrophy. Due to genetic variation between species, mouse models with mutations in the murine genes are of limited use to test and further optimize human specific AONs in vivo. To address this we have generated the del52hDMD/mdx mouse. This model carries both murine and human DMD genes. However, mouse dystrophin expression is abolished due to a stop mutation in exon 23, while the expression of human dystrophin is abolished due to a deletion of exon 52. The del52hDMD/mdx model, like mdx, shows signs of muscle dystrophy on a histological level and phenotypically mild functional impairment. Local administration of human specific vivo morpholinos induces exon skipping and dystrophin restoration in these mice. Depending on the number of mismatches, occasional skipping of the murine Dmd gene, albeit at low levels, could be observed. Unlike previous models, the del52hDMD/mdx model enables the in vivo analysis of human specific AONs targeting exon 51 or exon 53 on RNA and protein level and muscle quality and function. Therefore, it will be a valuable tool for optimizing human specific AONs and genome editing approaches for DMD.

Efficient and sensitive identification and quantification of airborne pollen using next-generation DNA sequencing.

Pollen monitoring is an important and widely used tool in allergy research and creation of awareness in pollen-allergic patients. Current pollen monitoring methods are microscope-based, labour intensive and cannot identify pollen to the genus level in some relevant allergenic plant groups. Therefore, a more efficient, cost-effective and sensitive method is needed. Here, we present a method for identification and quantification of airborne pollen using DNA sequencing. Pollen is collected from ambient air using standard techniques. DNA is extracted from the collected pollen, and a fragment of the chloroplast gene trnL is amplified using PCR. The PCR product is subsequently sequenced on a next-generation sequencing platform (Ion Torrent). Amplicon molecules are sequenced individually, allowing identification of different sequences from a mixed sample. We show that this method provides an accurate qualitative and quantitative view of the species composition of samples of airborne pollen grains. We also show that it correctly identifies the individual grass genera present in a mixed sample of grass pollen, which cannot be achieved using microscopic pollen identification. We conclude that our method is more efficient and sensitive than current pollen monitoring techniques and therefore has the potential to increase the throughput of pollen monitoring.

Loss-of-function mutations in MICU1 cause a brain and muscle disorder linked to primary alterations in mitochondrial calcium signaling.

Mitochondrial Ca(2+) uptake has key roles in cell life and death. Physiological Ca(2+) signaling regulates aerobic metabolism, whereas pathological Ca(2+) overload triggers cell death. Mitochondrial Ca(2+) uptake is mediated by the Ca(2+) uniporter complex in the inner mitochondrial membrane, which comprises MCU, a Ca(2+)-selective ion channel, and its regulator, MICU1. Here we report mutations of MICU1 in individuals with a disease phenotype characterized by proximal myopathy, learning difficulties and a progressive extrapyramidal movement disorder. In fibroblasts from subjects with MICU1 mutations, agonist-induced mitochondrial Ca(2+) uptake at low cytosolic Ca(2+) concentrations was increased, and cytosolic Ca(2+) signals were reduced. Although resting mitochondrial membrane potential was unchanged in MICU1-deficient cells, the mitochondrial network was severely fragmented. Whereas the pathophysiology of muscular dystrophy and the core myopathies involves abnormal mitochondrial Ca(2+) handling, the phenotype associated with MICU1 deficiency is caused by a primary defect in mitochondrial Ca(2+) signaling, demonstrating the crucial role of mitochondrial Ca(2+) uptake in humans.

Generation and characterization of transgenic mice with the full-length human DMD gene.

We report the generation of mice with an intact and functional copy of the 2.3-megabase human dystrophin gene (hDMD), the largest functional stretch of human DNA thus far integrated into a mouse chromosome. Yeast spheroplasts containing an artificial chromosome with the full-length hDMD gene were fused with mouse embryonic stem cells and were subsequently injected into mouse blastocysts to produce transgenic hDMD mice. Human-specific PCR, Southern blotting, and fluorescent in situ hybridization techniques demonstrated the intactness and stable chromosomal integration of the hDMD gene on mouse chromosome 5. Expression of the transgene was confirmed by RT-PCR and Western blotting. The tissue-specific expression pattern of the different DMD transcripts was maintained. However, the human Dp427p and Dp427m transcripts were expressed at 2-fold higher levels and human Dp427c and Dp260 transcripts were expressed at 2- and 4-fold lower levels than their endogenous counterparts. Ultimate functional proof of the hDMD transgene was obtained by crossing of hDMD mice with dystrophin-deficient mdx mice and dystrophin and utrophin-deficient mdx x Utrn-/- mice. The hDMD transgene rescued the lethal dystrophic phenotype of the mdx x Utrn-/- mice. All signs of muscular dystrophy disappeared in the rescued mice, as demonstrated by histological staining of muscle sections and gene expression profiling experiments. Currently, hDMD mice are extensively used for preclinical testing of sequence-specific therapeutics for the treatment of Duchenne muscular dystrophy. In addition, the hDMD mouse can be used to study the influence of the genomic context on deletion and recombination frequencies, genome stability, and gene expression regulation.

Targeted exon skipping in transgenic hDMD mice: A model for direct preclinical screening of human-specific antisense oligonucleotides.

The therapeutic potential of frame-restoring exon skipping by antisense oligonucleotides (AONs) has recently been demonstrated in cultured muscle cells from a series of Duchenne muscular dystrophy (DMD) patients. To facilitate clinical application, in vivo studies in animal models are required to develop safe and efficient AON-delivery methods. However, since exon skipping is a sequence-specific therapy, it is desirable to target the human DMD gene directly. We therefore set up human sequence-specific exon skipping in transgenic mice carrying the full-size human gene (hDMD). We initially compared the efficiency and toxicity of intramuscular AON injections using different delivery reagents in wild-type mice. At a dose of 3.6 nmol AON and using polyethylenimine, the skipping levels accumulated up to 3% in the second week postinjection and lasted for 4 weeks. We observed a correlation of this long-term effect with the intramuscular persistence of the AON. In regenerating myofibers higher efficiencies (up to 9%) could be obtained. Finally, using the optimized protocols in hDMD mice, we were able to induce the specific skipping of human DMD exons without affecting the endogenous mouse gene. These data highlight the high sequence specificity of this therapy and present the hDMD mouse as a unique model to optimize human-specific exon skipping in vivo.

Fluorescent labelling of cRNA for microarray applications.

Microarrays of oligonucleotide expression libraries can be hybridised with either cDNA, generated from mRNA during reverse transcription, or cRNA, generated in an Eberwine mRNA amplification procedure. While methods for fluorescent labelling of cDNA have been thoroughly investigated, methods for cRNA labelling have not. To this purpose, we developed an aminoallyl-UTP (aa-UTP) driven cRNA labelling protocol and compared it in expression profiling studies using spotted 7.5 K 65mer murine oligonucleotide arrays with labelling via direct incorporation of Cy-UTPs. The presence of dimethylsulfoxide during coupling of aa-modified cRNA with N-hydroxysuccinimide-modified, fluorescent Cy dyes greatly enhanced the labelling efficiency, as analysed by spectrophotometry and fluorescent hybridisation signals. Indirect labelling using aa-UTP resulted in 2- to 3-fold higher degrees of labelling and fluorescent signals than labelling by direct incorporation of Cy-UTP. By variation of the aa-UTP:UTP ratio, a clear optimal degree of labelling was found (1 dye per 20-25 nt). Incorporation of more label increased Cy3 signal but lowered Cy5 fluorescence. This effect is probably due to quenching, which is more prominent for Cy5 than for Cy3. In conclusion, the currently developed method is an efficient, robust and inexpensive technique for fluorescent labelling of cRNA and allows sensitive detection of gene expression profiles on oligonucleotide microarrays.