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Abstract
Traditional cloning methods have limitations on the number of DNA fragments that can be simultaneously manipulated, which dramatically slows the pace of molecular assembly. Here we describe GMAP, a Gibson assembly-based modular assembly platform consisting of a collection of promoters and genes, which allows for one-step production of DNA constructs. GMAP facilitates rapid assembly of expression and viral constructs using modular genetic components, as well as increasingly complicated genetic tools using contextually relevant genomic elements. Our data demonstrate the applicability of GMAP toward the validation of synthetic promoters, identification of potent RNAi constructs, establishment of inducible lentiviral systems, tumor initiation in genetically engineered mouse models, and gene-targeting for the generation of knock-in mice. GMAP represents a recombinant DNA technology designed for widespread circulation and easy adaptation for other uses, such as synthetic biology, genetic screens, and CRISPR-Cas9.
Highlights
Molecular cloning has allowed for manipulation of recombinant DNA to assemble DNA constructs that are widely used in molecular and synthetic biology
In order to assemble large genetic circuits of multiple transcriptional units, Guye et al designed a set of unique nucleotide sequences that when combined with Gateway Cloning and Gibson Assembly facilitate construction of a single construct used for transfection and stable integration in human cells[9]
Cells inducibly expressed short hairpin RNA (shRNA) and knockdown of the endogenous fluorescent protein was assessed by flow cytometry, identifying shRNA “C” as the most potent shRNA (Fig. 1f–h). These results demonstrate the ability of GMAP to rapidly assemble and functionally test in vitro retroviral and lentiviral constructs using standard genetic components such as synthetic promoters, tetracycline response elements, and shRNAs
Summary
Compatible backbones were created by designing gene blocks (gBlocks) from IDT (Supplementary Table S2) to clone into viral or R26TV constructs such that digestion and gel purification would yield linearized backbones with sites #1 and 5 or sites #2 and 5 terminal. The CAG-driven R26TV LSL backbone (R26TV CAG LSL 2−5) was created by cloning “Rosa[26] LSL 2–5 gBlock” into a R26TV LSL-GFP plasmid (Addgene plasmid 16103) linearized with Asc and XmaI such that digestion with PmeI eliminates a 389 bp spacer sequence between sites #2 and 5. This targeting construct has 5′ and 3′ homologous arms of 1.1 and 4.3 kb, respectively. Data are presented as mean ± s tandard error of the mean
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