Abstract
Random transgene integration is a powerful tool for developing new genome-wide screening approaches. These techniques have already been used for functional gene annotation by transposon-insertion sequencing, for identif ication of transcription factor binding sites and regulatory sequences, and for dissecting chromatin position effects. Precise localization of transgenes and accurate artifact f iltration are essential for this type of method. To date, many mapping assays have been developed, including Inverse-PCR, TLA, LAM-PCR, and splinkerette PCR. However, none of them is able to ensure localization of both transgene’s f lanking regions simultaneously, which would be necessary for some applications. Here we proposed a cheap and simple NGS-based approach that overcomes this limitation. The developed assay requires using intentionally designed vectors that lack recognition sites of one or a set of restriction enzymes used for DNA fragmentation. By looping and sequencing these DNA fragments, we obtain special data that allows us to link the two f lanking regions of the transposon. This can be useful for precise insertion mapping and for screening approaches in the f ield of chromosome engineering, where chromosomal recombination events between transgenes occur in a cell population. To demonstrate the method’s feasibility, we applied it for mapping SB transposon integration in the human HAP1 cell line. Our technique allowed us to eff iciently localize genomic transposon integrations, which was conf irmed via PCR analysis. For practical application of this approach, we proposed a set of recommendations and a normalization strategy. The developed method can be used for multiplex transgene localization and detection of rearrangements between them.
Highlights
Genome-wide screening assays are important tools for modern genetics and genomics
As in conventional inverse-PCR, our assay consists of five steps: 1) DNA fragmentation, 2) ligation under low DNA concentration conditions, which favors circularization, 3) nested-PCR using primer pairs annealing to the ends of transgenic sequen ce in outward orientation, 4) PCR products sequencing, and 5) computational analysis
In our modification (Fig. 1), we propose to fragment DNA by restriction enzyme (RE), which recognizes sites that are absent in the transgene sequence
Summary
Genome-wide screening assays are important tools for modern genetics and genomics Many of these methods rely on the integration of exogenous sequences in unknown or random genomic regions, mostly via retroviral or transposon vectors. This approach has already been used for functional gene annotation by transposon-insertion sequencing (Deutschbauer et al, 2011; Goodman et al, 2011; Goh et al, 2017; Cain et al, 2020), for transcription factor binding sites (Wang et al, 2012; Moudgil et al, 2020) and regulatory sequences identification (Pindyurin et al, 2015), and for chromatin position effects dissection (Akhtar et al, 2013). Our approach provides double-sided transgene locali zation that can be applied for translocation detection between transgene integration points
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
