A toolkit for assembly of targeting clones for C. elegans transgenesis.
A toolkit for assembly of targeting clones for C. elegans transgenesis.
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DOI：
10.17912/micropub.biology.000966
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发表时间：
2023
影响因子：
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通讯作者：
Nonet, Michael
中科院分区：
文献类型：
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作者：
Knoebel, Emma;Dour, Scott;Nonet, Michael
转基因蠕虫是线虫研究人员使用这种简单的后生动物模型系统解剖分子途径的关键资源。转基因提供了一种途径，可以使用遗传编码的传感器来可视化活动物的发育事件、细胞过程以及实时信号事件。随着转座子、CRISPR和重组酶介导的整合等多种整合方法的出现，这些工具的生成变得越来越高效。转基因生产的一个日益严重的限制是用于直接将序列插入基因组的靶向构建体的组装。在这里，我们提供了一个工具包，它使用基于金门(GG)克隆的方法来促进复杂记者的快速组装。使用进入质粒库，可以常规地高效地将一到八个DNA片段共同组装成整合载体。该工具包由20个SAPI GG进入载体和100个SAPI GG插入质粒组成，其中包含各种启动子、FP、标签、连接子、ORF、3‘UTRs和许多用于两部分表达系统的组件，这些组件可以混合在一起创建一个庞大的报告构建库。组装过程也可以很好地与聚合酶链式反应产物和5‘磷酸化的双链寡核苷酸一起工作，这样的DNA可以用来向管道中提供新的基因、启动子和标签。此外，该工具包还提供了一系列12个基于BsaI的空GG组装载体，以促进包含新插入的额外SAPI GG质粒的构建。作为附录和基于Microsoft®Excel的组装工具，提供了概述整个方法的手册，允许用户在组装模板的每个位置的克隆库中选择单个插入物，并输出带注释的序列。组装过程可以很容易地被多路复用，通常效率超过90%。这种方法的效率足以使显微注射而不是克隆产生成为转基因产生的限制因素。
Transgenic worms are a key resource for C. elegans researchers dissecting molecular pathways using this simple metazoan model system. Transgenes provide an avenue to visualize developmental events, cellular processes as well as real-time signal events in live animals using genetically encoded sensors. Generation of these tools has become increasingly efficient with the advent of numerous integration methods including transposon, CRISPR and recombinase-mediated integration. A growing limitation in transgene production is the assembly of the targeting constructs used to direct insertion of sequences into the genome. Here we present a toolkit that facilitates rapid assembly of complex reporters using a Golden Gate (GG) cloning-based approach. Co-assembly of one to eight DNA segments into an integration vector can be routinely obtained at high efficiency using a library of entry plasmids. The toolkit consists of 20 SapI GG entry vectors and 100 SapI GG insert plasmids containing a variety of promoters, FPs, tags, linkers, ORFs, 3' UTRs and numerous components for bipartite expression systems that can be mixed to create a huge repertoire of reporter constructs. The assembly process also works well with PCR products and 5' phosphorylated double stranded oligonucleotides, and such DNAs can be used to supply novel genes, promoters, and tags into the pipeline. In addition, the toolkit also provides a series of 12 empty BsaI -based GG assembly vectors that facilitate the construction of additional SapI GG plasmids containing novel inserts. A manual outlining the entire approach is provided as an appendix as well as a Microsoft® Excel based assembly tool which allows the user to choose individual inserts among the libraries of clones at each position in the assembly template and output an annotated sequence. The assembly process can easily be multiplexed and is typically over 90% efficient. The approach is sufficiently efficient to make microinjection rather than clone generation the limiting factor in transgene generation.