ABSTRACT
A core tenet of synthetic biology is that well-characterized regulatory elements are essential for engineering biological systems. Here, we characterize the specificity and expression levels of 18 short (254 to 880 bp) candidate germline promoters using a single-copy gfp reporter assay in C. elegans . Six promoters resulted in ubiquitous expression, three did not drive detectable expression, and nine were germline-specific. Several promoters drove stronger germline expression than the commonly-used mex-5 promoter. The promoters range across expression levels and facilitate, for example, low expression of toxic transgenes or high expression of gene editing enzymes, and their compactness facilitates gene synthesis.
ABSTRACT
Transgenes 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.
