King Abdullah University of Science and Technology (KAUST), Biological and Environmental Science and Engineering Division (BESE), KAUST Environmental Epigenetics Program (KEEP), Thuwal, 23955-6900, Saudi Arabia.
Nat Commun. 2020 Dec 9;11(1):6300. doi: 10.1038/s41467-020-19898-0.
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 A/T 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.
转基因为其结构、拷贝数和基因组位置容易发生渐进性沉默。在秀丽隐杆线虫中，抑制机制在生殖系中特别强，简单的染色体外阵列中的转基因完全沉默，单拷贝转基因插入的频繁沉默。一类非编码 DNA，周期性 A/T 簇（PATC）可以防止抑制性染色质或小干扰 RNA（piRNA）中的转基因沉默。在这里，我们描述了从野生型动物阵列中有效进行生殖系表达的设计规则（密码子优化、内含子和 PATC 包含、高温（25°C）和载体骨架去除）。我们生成了基于网络的工具来分析 PATC 并提供方便组装富含 PATC 的转基因的试剂。广泛的沉默抗性荧光蛋白（例如 GFP、mCherry 和 tagBFP）可用于剖析生殖系调控元件，一组增强酶（Mos1 转座酶、Cas9、Cre 和 Flp 重组酶）可在秀丽隐杆线虫中实现高效的基因工程。
