Abstract
AbstractTransgenes are prone to progressive silencing due to their structure, copy number, and genomic location. In C. elegans, repressive mechanisms are particularly strong in the germline with almost fully penetrant transgene silencing in simple extrachromosomal arrays and frequent silencing of single-copy transgene insertions. A class of non-coding DNA, Periodic An/Tn Clusters (PATCs) can prevent transgene-silencing in repressive chromatin or from small interfering RNAs (piRNAs). Here, we describe design rules (codon-optimization, intron and PATC inclusion, elevated temperature (25 °C), and vector backbone removal) for efficient germline expression from arrays in wildtype animals. We generate web-based tools to analyze PATCs and reagents for the convenient assembly of PATC-rich transgenes. An extensive collection of silencing resistant fluorescent proteins (e.g., gfp, mCherry, and tagBFP) can be used for dissecting germline regulatory elements and a set of enhanced enzymes (Mos1 transposase, Cas9, Cre, and Flp recombinases) enable efficient genetic engineering in C. elegans.
| Original language | English (US) |
|---|---|
| Journal | Nature Communications |
| Volume | 11 |
| Issue number | 1 |
| DOIs | |
| State | Published - Dec 9 2020 |
Bibliographical note
KAUST Repository Item: Exported on 2020-12-17Acknowledgements: We thank Andrew Z. Fire and Erik M. Jorgensen for experimental support, M. Wayne Davis for bioinformatic assistance, and Kam Hoe for technical assistance. Some strains were provided by the CGC, which is funded by NIH Office of Research Infrastructure Programs (P40 OD010440). We thank the KAUST Bioscience Core Labs and the Linux and Advanced Platforms team for expert assistance and Faisal Alkhaldi for assistance developing www.wormbuilder.org. This work was funded by a KAUST intramural grant and the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.