利用 Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS) 进行 中的高通量文库转基因

High-throughput library transgenesis in via Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS).

机构信息

Institute of Ecology and Evolution, University of Oregon, Eugene, United States.

School of Chemical & Biomolecular Engineering, Georgia Institute of Technology, Atlanta, United States.

出版信息

Elife. 2023 Jul 4;12:RP84831. doi: 10.7554/eLife.84831.

Abstract

High-throughput transgenesis using synthetic DNA libraries is a powerful method for systematically exploring genetic function. Diverse synthesized libraries have been used for protein engineering, identification of protein-protein interactions, characterization of promoter libraries, developmental and evolutionary lineage tracking, and various other exploratory assays. However, the need for library transgenesis has effectively restricted these approaches to single-cell models. Here, we present Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS), a simple yet powerful approach to large-scale transgenesis that overcomes typical limitations encountered in multicellular systems. TARDIS splits the transgenesis process into a two-step process: creation of individuals carrying experimentally introduced sequence libraries, followed by inducible extraction and integration of individual sequences/library components from the larger library cassette into engineered genomic sites. Thus, transformation of a single individual, followed by lineage expansion and functional transgenesis, gives rise to thousands of genetically unique transgenic individuals. We demonstrate the power of this system using engineered, split selectable TARDIS sites in to generate (1) a large set of individually barcoded lineages and (2) transcriptional reporter lines from predefined promoter libraries. We find that this approach increases transformation yields up to approximately 1000-fold over current single-step methods. While we demonstrate the utility of TARDIS using , in principle the process is adaptable to any system where experimentally generated genomic loci landing pads and diverse, heritable DNA elements can be generated.

摘要

使用合成 DNA 文库进行高通量转基因是系统探索遗传功能的有力方法。各种合成文库已被用于蛋白质工程、蛋白质-蛋白质相互作用的鉴定、启动子文库的表征、发育和进化谱系追踪以及各种其他探索性测定。然而,文库转基因的需求有效地将这些方法限制在单细胞模型中。在这里,我们提出了 Transgenic Arrays Resulting in Diversity of Integrated Sequences (TARDIS),这是一种简单而强大的大规模转基因方法,克服了多细胞系统中遇到的典型限制。TARDIS 将转基因过程分为两步:创建携带实验引入序列文库的个体,然后从较大的文库盒中诱导提取和整合个体序列/文库组件到工程化基因组位点。因此,单个个体的转化, followed by 谱系扩张和功能转基因,产生数千个具有遗传独特性的转基因个体。我们使用 中的工程化、分裂可选择的 TARDIS 位点来证明该系统的强大功能,以生成 (1) 一组大量单独标记的谱系和 (2) 来自预定义启动子文库的转录报告基因系。我们发现,与当前的单步方法相比,这种方法将转化产量提高了约 1000 倍。虽然我们使用 证明了 TARDIS 的实用性,但原则上,该过程适用于任何可以生成实验生成的基因组着陆点和多样化、可遗传 DNA 元件的系统。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5536/10328503/cd1f7cd30496/elife-84831-fig1.jpg

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