Abstract
The cellular slime mold Dictyostelium discoideum is a powerful model organism that can be utilized to investigate human health and disease. One particular strength of Dictyostelium is that it can be utilized for high throughput genetic screens. For many phenotypes, one limitation of utilizing Dictyostelium is that screening can be an arduous and time-consuming process, limiting the genomic depth one can cover. Previously, we utilized a restriction enzyme-mediated integration screen to identify suppressors of polyglutamine aggregation in Dictyostelium. However, due to the time required to perform the screen, we only obtained ∼4% genome coverage. Here we have developed an efficient screening pipeline that couples chemical mutagenesis with the 5-fluoroorotic acid counterselection system to enrich for mutations in genes of interest. Here we describe this new screening methodology and highlight how it can be utilized for other biological systems.
Highlights
Model organisms like Dictyostelium discoideum (Dictyostelium) offer numerous advantages for identifying key components of cellular systems
5-FOA treated cells when compared to the DMSO control which contains a majority of cells with diffuse GFP
Samples were analyzed by an unpaired t-test, ∗∗∗ indicates p = 0.0007
Summary
Model organisms like Dictyostelium discoideum (Dictyostelium) offer numerous advantages for identifying key components of cellular systems. Restriction enzyme-mediated integration (REMI) enables screens that rely on disrupting gene function via integration of plasmid into the Dictyostelium genome (Guerin and Larochelle, 2002). Affected genes can be identified by extracting the plasmid from the genome with restriction enzymes and sequencing retained pieces of genomic DNA to identify disrupted genes (Guerin and Larochelle, 2002). Mutagenesis using a chemical mutagen such as N-methyl-N -nitro-N-nitroguanidine (NTG) can introduce mutations into the Dictyostelium genome that can later be identified by whole-genome sequencing (Li et al, 2016). With these assays, one can rapidly generate mutations at near genome saturation. Developing methods to positively select for phenotypes of interest is a powerful way to increase the depth of genomic coverage
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