IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-t... more IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in tm1830 mutants. ds distal segment, ms middle segment, bb basal body region, den dendrite. All images are similarly scaled and orientated (arrow denotes basal body). Scale bar, 3 μm. c Representative kymographs (time (t) over distance (d) plots) used to generate IFT rate measurements. For each kymo...
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane prote... more Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP 5 and M and the minor proteins GP 2a , E, GP 3 , and GP 4 . Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP 5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indi...
Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as ... more Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.
Supplementary information to the data in Fig. 8 . a Schematic representation of all the differen... more Supplementary information to the data in Fig. 8 . a Schematic representation of all the different KIAA0556 fragments used to screen our selection of 200+ ciliary proteins. The predicted protein repeat domains, shown in Additional files 1 and 2, are depicted as d1 to d4. Constructs were generated containing isolated domains as well as a combination of domains. b Single transfections of PalMyr-KIAA0556 and mRFP-KATNBL1, showing that membrane localisation of the mRFP tagged protein is indeed dependent on the interaction with the PalMyr-tagged protein. (JPG 491 kb)
Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn er... more Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5’-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,...
Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigm... more Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G>T, both reside in exon 13. Skipping of exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection, and induced a detectable level of exon skipping until at least 6 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic appro... more Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of humanmutations affecting pre-mRNAsplicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy.
Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are... more Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are fluorescence images of worms expressing a transcriptional GFP reporter under the control of the indicated geneâ s promoter, which stains the entire cell in which it is expressed. DiI (red) co-stain identifies six pairs of ciliated amphid neurons and both pairs of ciliated phasmid neurons. Arrowheads denote cells with both red and green signals. Other ciliated head cells are identifiable by long dendritic processes (arrows) extending to the anterior end of the worm. Scale bars, 20Â Îźm (all images similarly scaled). (JPG 611 kb)
IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-t... more IFT analysis in C. elegans K04F10.2( tm1830 ) mutants. a Intraflagellar transport rates in wild-type and K04F10.2(tm1830) mutant worms. Shown are the anterograde and retrograde velocities (μm.s-1/standard deviation (SD)) of GFP-tagged IFT proteins along amphid and phasmid channel cilia (combined; top rows), or phasmid cilia only (bottom rows). t-test pairwise comparison with wild-type controls, n number of particles, N measured number of amphids and phasmids. OSM-3 is the worm orthologue of KIF17; CHE-11 is the worm orthologue of IFT140; OSM-6 is the worm orthologue of IFT52. b Representative fluorescence images of phasmid cilia showing normal IFT protein localisations and distributions in tm1830 mutants. ds distal segment, ms middle segment, bb basal body region, den dendrite. All images are similarly scaled and orientated (arrow denotes basal body). Scale bar, 3 μm. c Representative kymographs (time (t) over distance (d) plots) used to generate IFT rate measurements. For each kymo...
Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane prote... more Virions of porcine reproductive and respiratory syndrome virus (PRRSV) contain six membrane proteins: the major proteins GP 5 and M and the minor proteins GP 2a , E, GP 3 , and GP 4 . Here, we studied the envelope protein requirements for PRRSV particle formation and infectivity using full-length cDNA clones in which the genes encoding the membrane proteins were disrupted by site-directed mutagenesis. By transfection of RNAs transcribed from these cDNAs into BHK-21 cells and analysis of the culture medium using ultracentrifugation, radioimmunoprecipitation, and real-time reverse transcription-PCR, we observed that the production of viral particles is dependent on both major envelope proteins; no particles were released when either the GP 5 or the M protein was absent. In contrast, particle production was not dependent on the minor envelope proteins. Remarkably, in the absence of any one of the latter proteins, the incorporation of all other minor envelope proteins was affected, indi...
Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as ... more Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.
Supplementary information to the data in Fig. 8 . a Schematic representation of all the differen... more Supplementary information to the data in Fig. 8 . a Schematic representation of all the different KIAA0556 fragments used to screen our selection of 200+ ciliary proteins. The predicted protein repeat domains, shown in Additional files 1 and 2, are depicted as d1 to d4. Constructs were generated containing isolated domains as well as a combination of domains. b Single transfections of PalMyr-KIAA0556 and mRFP-KATNBL1, showing that membrane localisation of the mRFP tagged protein is indeed dependent on the interaction with the PalMyr-tagged protein. (JPG 491 kb)
Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn er... more Pyridoxine-dependent epilepsy (PDE-ALDH7A1), also known as antiquitin deficiency, is an inborn error of lysine metabolism that presents with refractory epilepsy in newborns. Bi-allelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important co-factor pyridoxal-5’-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but despite this treatment, intellectual disability may occur. Early diagnosis and treatment, preferably based on newborn screening, potentially optimize long-term clinical outcome. However, the currently known diagnostic PDE-ALDH7A1 biomarkers are incompatible with newborn screening procedures. Using a combination of the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy, we have been able to discover novel biomarkers for PDE-ALDH7A1: 2S,...
Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigm... more Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G>T, both reside in exon 13. Skipping of exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in iPSC-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection, and induced a detectable level of exon skipping until at least 6 months post injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.
Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic appro... more Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of humanmutations affecting pre-mRNAsplicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy.
Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are... more Post-embryonic tissue expression of C. elegans katanin genes mei-1 , mei-2 and F47G4.4. Shown are fluorescence images of worms expressing a transcriptional GFP reporter under the control of the indicated geneâ s promoter, which stains the entire cell in which it is expressed. DiI (red) co-stain identifies six pairs of ciliated amphid neurons and both pairs of ciliated phasmid neurons. Arrowheads denote cells with both red and green signals. Other ciliated head cells are identifiable by long dendritic processes (arrows) extending to the anterior end of the worm. Scale bars, 20Â Îźm (all images similarly scaled). (JPG 611 kb)
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