A Simple and Scalable Strategy for Analysis of Endogenous Protein Dynamics.

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically, it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose, a large set of proteins representing diverse structures and functions, some of which are known or potential drug targets, were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86% of the targets, as determined by luminescence-based plate assays, blotting, and imaging. In order to determine whether endogenously tagged proteins yield more representative models, cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases, only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches.

Leukemogenesis in heterozygous PU.1 knockout mice.

Most murine radiation-induced acute myeloid leukemias involve biallelic inactivation of the PU.1 gene, with one allele being lost through a radiation-induced chromosomal deletion and the other allele affected by a recurrent point mutation in codon 235 that is likely to be spontaneous. The short latencies of acute myeloid leukemias occurring in nonirradiated mice engineered with PU.1 conditional knockout or knockdown alleles suggest that once both copies of PU.1 have been lost any other steps involved in leukemogenesis occur rapidly. Yet, spontaneous acute myeloid leukemias have not been reported in mice heterozygous for a PU.1 knockout allele, an observation that conflicts with the understanding that the PU.1 codon 235 mutation is spontaneous. Here we describe experiments that show that the lack of spontaneous leukemia in PU.1 heterozygous knockout mice is not due to insufficient monitoring times or mouse numbers or the genetic background of the knockout mice. The results reveal that spontaneous leukemias that develop in mice of the mixed 129S2/SvPas and C57BL/6 background of knockout mice arise by a pathway that does not involve biallelic PU.1 mutation. In addition, the latency of radiation-induced leukemia in PU.1 heterozygous mice on a genetic background susceptible to radiation-induced leukemia indicates that the codon 235 mutation is not a rate-limiting step in radiation leukemogenesis driven by PU.1 loss.

Molecular characterisation of murine acute myeloid leukaemia induced by 56Fe ion and 137Cs gamma ray irradiation.

Exposure to sparsely ionising gamma- or X-ray irradiation is known to increase the risk of leukaemia in humans. However, heavy ion radiotherapy and extended space exploration will expose humans to densely ionising high linear energy transfer (LET) radiation for which there is currently no understanding of leukaemia risk. Murine models have implicated chromosomal deletion that includes the hematopoietic transcription factor gene, PU.1 (Sfpi1), and point mutation of the second PU.1 allele as the primary cause of low-LET radiation-induced murine acute myeloid leukaemia (rAML). Using array comparative genomic hybridisation, fluorescence in situ hybridisation and high resolution melt analysis, we have confirmed that biallelic PU.1 mutations are common in low-LET rAML, occurring in 88% of samples. Biallelic PU.1 mutations were also detected in the majority of high-LET rAML samples. Microsatellite instability was identified in 42% of all rAML samples, and 89% of samples carried increased microsatellite mutant frequencies at the single-cell level, indicative of ongoing instability. Instability was also observed cytogenetically as a 2-fold increase in chromatid-type aberrations. These data highlight the similarities in molecular characteristics of high-LET and low-LET rAML and confirm the presence of ongoing chromosomal and microsatellite instability in murine rAML.

Genetic isolation and characterization of a splicing mutant of zebrafish dystrophin.

Sapje-like (sap(cl100)) was one of eight potential zebrafish muscle mutants isolated as part of an early-pressure screen of 500 families. This mutant shows a muscle tearing phenotype similar to sapje (dys-/-) and both mutants fail to genetically complement suggesting they have a mutation in the same gene. Protein analysis confirms a lack of dystrophin in developing sapje-like embryos. Sequence analysis of the sapje-like dystrophin mRNA shows that exon 62 is missing in the dystrophin transcript causing exon 63 to be translated out of frame terminating translation at a premature stop codon at the end of exon 63. Sequence analysis of sapje-like genomic DNA identified a mutation in the donor splice junction at the end of dystrophin exon 62. This mutation is similar to splicing mutations associated with human forms of Duchenne Muscular Dystrophy. Sapje-like is the first zebrafish dystrophin splicing mutant identified to date and represents a novel disease model which can be used in future studies to identify therapeutic compounds for treating diseases caused by splicing defects.

The zebrafish runzel muscular dystrophy is linked to the titin gene.

Titin (also called connectin) acts as a scaffold for signaling proteins in muscle and is responsible for establishing and maintaining the structure and elasticity of sarcomeres in striated muscle. Several human muscular dystrophies and cardiomyopathies have previously been linked to mutations in the titin gene. This study reports linkage of the runzel homozygous lethal muscular dystrophy in the zebrafish Danio rerio to a genomic interval containing the titin gene. Analysis of the genomic sequence suggests that zebrafish contain two adjacent titin loci. One titin locus lies within the genetic linkage interval and its expression is significantly reduced in runzel mutants by both immunofluorescence and protein electrophoresis. Morpholino downregulation of this same titin locus in wild-type embryos results in decreased muscle organization and mobility, phenocopying runzel mutants. Additional protein analysis demonstrates that, in wild-type zebrafish, titin isoform sizes are rapidly altered during the development of striated muscle, likely requiring a previously unrecognized need for vertebrate sarcomere remodeling to incorporate developmentally regulated titin isoforms. Decreases of affected titin isoforms in runzel mutants during this time correlate with a progressive loss of sarcomeric organization and suggest that the unaffected titin proteins are capable of sarcomerogenesis but not sarcomere maintenance. In addition, microarray analysis of the ruz transcriptome suggests a novel mechanism of dystrophy pathogenesis, involving mild increases in calpain-3 expression and upregulation of heat shock proteins. These studies should lead to a better understanding of titin's role in normal and diseased muscle.

Zebrafish orthologs of human muscular dystrophy genes.

BACKGROUND: Human muscular dystrophies are a heterogeneous group of genetic disorders which cause decreased muscle strength and often result in premature death. There is no known cure for muscular dystrophy, nor have all causative genes been identified. Recent work in the small vertebrate zebrafish Danio rerio suggests that mutation or misregulation of zebrafish dystrophy orthologs can also cause muscular degeneration phenotypes in fish. To aid in the identification of new causative genes, this study identifies and maps zebrafish orthologs for all known human muscular dystrophy genes. RESULTS: Zebrafish sequence databases were queried for transcripts orthologous to human dystrophy-causing genes, identifying transcripts for 28 out of 29 genes of interest. In addition, the genomic locations of all 29 genes have been found, allowing rapid candidate gene discovery during genetic mapping of zebrafish dystrophy mutants. 19 genes show conservation of syntenic relationships with humans and at least two genes appear to be duplicated in zebrafish. Significant sequence coverage on one or more BAC clone(s) was also identified for 24 of the genes to provide better local sequence information and easy updating of genomic locations as the zebrafish genome assembly continues to evolve. CONCLUSION: This resource supports zebrafish as a dystrophy model, suggesting maintenance of all known dystrophy-associated genes in the zebrafish genome. Coupled with the ability to conduct genetic screens and small molecule screens, zebrafish are thus an attractive model organism for isolating new dystrophy-causing genes/pathways and for use in high-throughput therapeutic discovery.

Modeling human muscle disease in zebrafish.

Zebrafish reproduce in large quantities, grow rapidly, and are transparent early in development. For these reasons, zebrafish have been used extensively to model vertebrate development and disease. Like mammals, zebrafish express dystrophin and many of its associated proteins early in development and these proteins have been shown to be vital for zebrafish muscle stability. In dystrophin-null zebrafish, muscle degeneration becomes apparent as early as 3 days post-fertilization (dpf) making the zebrafish an excellent organism for large-scale screens to identify other genes involved in the disease process or drugs capable of correcting the disease phenotype. Being transparent, developing zebrafish are also an ideal experimental model for monitoring the fate of labeled transplanted cells. Although zebrafish dystrophy models are not meant to replace existing mammalian models of disease, experiments requiring large numbers of animals may be best performed in zebrafish. Results garnered from using this model could lead to a better understanding of the pathogenesis of the muscular dystrophies and the development of future therapies.

Delta-sarcoglycan is required for early zebrafish muscle organization.

Mutations in sarcoglycans (alpha-, beta-, gamma-, and delta-) have been linked with limb girdle muscular dystrophy (LGMD) types 2C-F in humans. We have cloned the zebrafish orthologue encoding delta-sarcoglycan and mapped the gene to linkage group 21. The predicted zebrafish delta-sarcoglycan protein is highly homologous with its human orthologue including conservation of two of the three predicted glycosylation sites. Like other members of the dystrophin-associated protein complex (DAPC), delta-sarcoglycan localizes to the sarcolemmal membrane of the myofiber in adult zebrafish, but is more apparent at the myosepta in developing embryos. Zebrafish embryos injected with morpholinos against delta-sarcoglycan were relatively inactive at 5 dpf, their myofibers were disorganized, and swim bladders uninflated. Immunohistochemical and immunoblotting experiments show that delta-, beta-, and gamma-sarcoglycans were all downregulated in the morphants, whereas dystrophin expression was unaffected. Whereas humans lacking delta-sarcoglycan primarily show adult phenotypes, our results suggest that delta-sarcoglycan plays a role in early zebrafish muscle development.