20 research outputs found
Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila
Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. Here we describe a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNA^Δ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNA^Δ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNA^Δ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved
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A drug-inducible sex-separation technique for insects
Here, we describe a drug-inducible genetic system for insect sex-separation that demonstrates proof-of-principle for positive sex selection in D. melanogaster. The system exploits the toxicity of commonly used broad-spectrum antibiotics geneticin and puromycin to kill the non-rescued sex. Sex-specific rescue is achieved by inserting sex-specific introns into the coding sequences of antibiotic-resistance genes. When raised on geneticin-supplemented food, the sex-sorter line establishes 100% positive selection for female progeny, while the food supplemented with puromycin positively selects 100% male progeny. Since the described system exploits conserved sex-specific splicing mechanisms and reagents, it has the potential to be adaptable to other insect species of medical and agricultural importance
Selective removal of deletion-bearing mitochondrial DNA in heteroplasmic Drosophila
Mitochondrial DNA (mtDNA) often exists in a state of heteroplasmy, in which mutant mtDNA co-exists in cells with wild-type mtDNA. High frequencies of pathogenic mtDNA result in maternally inherited diseases; maternally and somatically acquired mutations also accumulate over time and contribute to diseases of ageing. Reducing heteroplasmy is therefore a therapeutic goal and in vivo models in post-mitotic tissues are needed to facilitate these studies. Here we describe a transgene-based model of a heteroplasmic lethal mtDNA deletion (mtDNA^Δ) in adult Drosophila muscle. Stimulation of autophagy, activation of the PINK1/parkin pathway or decreased levels of mitofusin result in a selective decrease in mtDNA^Δ. Decreased levels of mitofusin and increased levels of ATPIF1, an inhibitor of ATP synthase reversal-dependent mitochondrial repolarization, result in a further decrease in mtDNA^Δ levels. These results show that an adult post-mitotic tissue can be cleansed of a deleterious genome, suggesting that therapeutic removal of mutant mtDNA can be achieved
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Temperature-Inducible Precision-Guided Sterile Insect Technique
Releases of sterile males are the gold standard for many insect population control programs, and precise sex sorting to remove females prior to male releases is essential to the success of these operations. To advance traditional methods for scaling the generation of sterile males, we previously described a CRISPR-mediated precision-guided sterile insect technique (pgSIT), in which Cas9 and gRNA strains are genetically crossed to generate sterile males for mass release. While effective at generating F1 sterile males, pgSIT requires a genetic cross between the two parental strains, which requires maintenance and sexing of two strains in a factory. Therefore, to advance pgSIT further by removing this crossing step, here we describe a next-generation temperature-inducible pgSIT (TI-pgSIT) technology and demonstrate its proof-of-concept in Drosophila melanogaster. Importantly, we were able to develop a true breeding strain for TI-pgSIT that eliminates the requirement for sex sorting-a feature that may help further automate production at scale