Genetic analysis relies on the ability to introduce, eliminate or modify genes at will. Such techniques are advanced in genetic model organisms such as yeast, flies and mice, but are limited in the worm C. elegans, which is one of the most commonly used model organisms. In worms such methods are somewhat crude: gene knockouts are by random mutagenesis and restoring gene function is via multicopy extrachromosomal arrays or random gene integrations. Despite these limitations C. elegans research has contributed to some of the most important discoveries in biology in the last 35 years: Ras - MAP kinase pathways, cell death pathways, RNA interference and posttranscriptional regulation by microRNAs are examples. We propose to develop techniques to insert, delete or modify genes in the C. elegans genome. The techniques rely on mobilizing transposons to engineer the genome;the methods will be a resource for the whole C. elegans research community.
Aim 1. Improved single copy insertion. We will characterize insertion sites on each chromosome and increase the efficiency of transgene insertions. We will also devise transient selection reagents that can be used in a wild-type background.
Aim 2. Universal insertion sites. We will generate universal insertion sites on all chromosomes that will be compatible with a single targeting plasmid. This will substantially increase the versatility of the technique.
Aim 3. Gene targeting. We will develop a strategy to manipulate genes in their endogenous context. This technique will allow researchers to engineer mutations, including knock-outs, without any extraneous DNA changes in the gene except the intended mutation.

Public Health Relevance

C. elegans is one of the major model organisms for studies of cell biology;more than 300 C. elegans labs in the US currently receive funding from the NIH. These labs pursue projects studying genes involved in aging, cancer, toxicology, neuroscience, and response to pathogens. The ability to modify the C. elegans genome without limits will make experimentation much faster and results more reliable for studies of all aspects of human health.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Genomics, Computational Biology and Technology Study Section (GCAT)
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Janes, Daniel E
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University of Utah
Schools of Arts and Sciences
Salt Lake City
United States
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Frøkjær-Jensen, Christian; Jain, Nimit; Hansen, Loren et al. (2016) An Abundant Class of Non-coding DNA Can Prevent Stochastic Gene Silencing in the C. elegans Germline. Cell 166:343-357
Schwartz, Matthew L; Jorgensen, Erik M (2016) SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans. Genetics 202:1277-88
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Edelman, Theresa L B; McCulloch, Katherine A; Barr, Angela et al. (2016) Analysis of a lin-42/period Null Allele Implicates All Three Isoforms in Regulation of Caenorhabditis elegans Molting and Developmental Timing. G3 (Bethesda) 6:4077-4086
Frøkjær-Jensen, Christian (2015) Transposon-Assisted Genetic Engineering with Mos1-Mediated Single-Copy Insertion (MosSCI). Methods Mol Biol 1327:49-58
Frøkjær-Jensen, Christian; Davis, M Wayne; Sarov, Mihail et al. (2014) Random and targeted transgene insertion in Caenorhabditis elegans using a modified Mos1 transposon. Nat Methods 11:529-34
Frøkjær-Jensen, Christian (2013) Exciting prospects for precise engineering of Caenorhabditis elegans genomes with CRISPR/Cas9. Genetics 195:635-42
Frøkjær-Jensen, Christian; Davis, M Wayne; Ailion, Michael et al. (2012) Improved Mos1-mediated transgenesis in C. elegans. Nat Methods 9:117-8
Zeiser, Eva; Frøkjær-Jensen, Christian; Jorgensen, Erik et al. (2011) MosSCI and gateway compatible plasmid toolkit for constitutive and inducible expression of transgenes in the C. elegans germline. PLoS One 6:e20082