2022
DOI: 10.1101/2022.01.17.476579
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piggyBac-Mediated Genomic Integration of Linear dsDNA-Based Library for Deep Mutational Scanning in Mammalian Cells

Abstract: There is a need of introducing large variant libraries into mammalian cells to facilitate deep mutational scanning (DMS). In this study, we develop an approach to integrate in-vitro ligation dsDNA variant library into cultured mammalian cell genome via the piggyBac transposon system. T4 DNA ligase was employed to produce dsDNA ligation products containing variant library sequence flanked by piggyBac inverted terminal repeat sequences (ITRs) at both ends. The ligation products were then cotransfected with a pig… Show more

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Cited by 1 publication
(2 citation statements)
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References 60 publications
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“…This strategy takes advantages of both the large size of in vitro ligation products and the ideal genomic integration efficiency of PCR products and completely avoids the library size shrinkage in the preparation process of plasmid libraries. And we have demonstrated the feasibility of this strategy by the identification of green fluorescence-chromophore amino acid sequences and a transcriptional activation peptide …”
Section: Current Strategies To Construct Mammalian Cell-based Dna-enc...mentioning
confidence: 99%
See 1 more Smart Citation
“…This strategy takes advantages of both the large size of in vitro ligation products and the ideal genomic integration efficiency of PCR products and completely avoids the library size shrinkage in the preparation process of plasmid libraries. And we have demonstrated the feasibility of this strategy by the identification of green fluorescence-chromophore amino acid sequences and a transcriptional activation peptide …”
Section: Current Strategies To Construct Mammalian Cell-based Dna-enc...mentioning
confidence: 99%
“…And we have demonstrated the feasibility of this strategy by the identification of green fluorescence-chromophore amino acid sequences and a transcriptional activation peptide. 45 2.2.2. Targeted Genomic Integration of the Libraries.…”
Section: Construction Of Genome-integrated Libraries In Mammalian Cellsmentioning
confidence: 99%