Wild type (WT) full length POLQ cDNA was assembled by PCR with a FLAG-P2A-BLAST cassette and subsequently cloned by Gibson assembly (New England BioLabs, #E2611L) in the PiggyBac PBCAG-eGFP vector (Addgene #40973) using BsrGI and SbfI sites to obtain the PiggyBac-eGFP-POLQ-FLAG-P2A-BLAST vector (referred here as GFP-Polθ WT). Point mutations were introduced using PCR mutagenesis and Gibson assembly in active sites as follow: Helicase (K121A, DE-216/7-AA), polymerase (DE-2540/1-AA), helicase/polymerase (K121A, DE-216/7-AA, DE-2540/1-AA). RPE1b POLQ−/− cells were complemented with GFP-Polθ WT and mutant constructs. Cells were plated in a 6-well plate, transfected the day after with 1 μg of PiggyBac vector and 0.5 μg of PiggyBac transposase (System Biosciences, #PB210PA-1) using Lipofectamine LTX with Plus Reagent (Thermo Fisher) and selected with Blasticidin (21 μg/ml) for 10 days. Vectors expression was confirmed by Western Blot.
Piggybac pbcag egfp vector
Manufactured by Addgene
The PiggyBac PBCAG-eGFP vector is a plasmid that contains the PiggyBac transposon system and an enhanced green fluorescent protein (eGFP) gene. The PiggyBac system is a tool used for genomic integration, while the eGFP gene allows for the visualization of transfected cells.
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2 protocols using piggybac pbcag egfp vector
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Engineered POLQ Mutants in RPE1b Cells
2
Engineered POLQ Mutants in RPE1b Cells
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Wild type (WT) full length POLQ cDNA was assembled by PCR with a FLAG-P2A-BLAST cassette and subsequently cloned by Gibson assembly (New England BioLabs, #E2611L) in the PiggyBac PBCAG-eGFP vector (Addgene #40973) using BsrGI and SbfI sites to obtain the PiggyBac-eGFP-POLQ-FLAG-P2A-BLAST vector (referred here as GFP-Polθ WT). Point mutations were introduced using PCR mutagenesis and Gibson assembly in active sites as follow: Helicase (K121A, DE-216/7-AA), polymerase (DE-2540/1-AA), helicase/polymerase (K121A, DE-216/7-AA, DE-2540/1-AA). RPE1b POLQ−/− cells were complemented with GFP-Polθ WT and mutant constructs. Cells were plated in a 6-well plate, transfected the day after with 1 μg of PiggyBac vector and 0.5 μg of PiggyBac transposase (System Biosciences, #PB210PA-1) using Lipofectamine LTX with Plus Reagent (Thermo Fisher) and selected with Blasticidin (21 μg/ml) for 10 days. Vectors expression was confirmed by Western Blot.
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