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Evaluation of PiggyBac transposon integration. A, Time course for evaluation of PiggyBac transposon (PB transposon) integration. The day when the donor vector carrying EGFP flanked by PiggyBac inverted terminal repeats (ITRs) and the effector vector carrying mammalian codon-optimised PiggyBac transposase (mPBase) were transfected at several concentrations was defined as Day 0. Fluorescence-activated cell sorting (FACS) was conducted every 2 days after transfection to confirm the ration of EGFP-positive mES cells. Passages were conducted using the remaining murine embryonic stem (mES) cells after FACS. B, Ratio of EGFP-positive mES cells and days after transfection. EGFP-positive mES cells were calculated among a total of 200 mES cells passing quality filters by FACS. Blank symbols in the left figure indicate mES cells transfected with 200 ng of donor vector, and filled symbols in the right figure indicate those transfected with 2,000 ng of donor vector. Gray circles, black squares, blue diamonds, and orange triangles indicate mES cells transfected with 0, 350, 3500, and 17500 ng of effector vector, respectively. We stopped evaluating mES cells transfected with a very high concentration (17500 ng) of effector vector on Day 4. C, Histogram of EGFP-positive mES cells on Day 10. The upper table shows the conditions of transfection. Lower histograms show all EGFP-positive mES cells in a total of 200 mES cells passing quality filters. The x-axis shows EGFP signal intensity, and the y-axis shows counts of EGFP-positive mES cells.

Journal: bioRxiv

Article Title: Regional random mutagenesis driven by multiple sgRNAs and diverse on-target genome editing events to identify functionally important elements in non-coding regions

doi: 10.1101/2023.09.12.557308

Figure Lengend Snippet: Evaluation of PiggyBac transposon integration. A, Time course for evaluation of PiggyBac transposon (PB transposon) integration. The day when the donor vector carrying EGFP flanked by PiggyBac inverted terminal repeats (ITRs) and the effector vector carrying mammalian codon-optimised PiggyBac transposase (mPBase) were transfected at several concentrations was defined as Day 0. Fluorescence-activated cell sorting (FACS) was conducted every 2 days after transfection to confirm the ration of EGFP-positive mES cells. Passages were conducted using the remaining murine embryonic stem (mES) cells after FACS. B, Ratio of EGFP-positive mES cells and days after transfection. EGFP-positive mES cells were calculated among a total of 200 mES cells passing quality filters by FACS. Blank symbols in the left figure indicate mES cells transfected with 200 ng of donor vector, and filled symbols in the right figure indicate those transfected with 2,000 ng of donor vector. Gray circles, black squares, blue diamonds, and orange triangles indicate mES cells transfected with 0, 350, 3500, and 17500 ng of effector vector, respectively. We stopped evaluating mES cells transfected with a very high concentration (17500 ng) of effector vector on Day 4. C, Histogram of EGFP-positive mES cells on Day 10. The upper table shows the conditions of transfection. Lower histograms show all EGFP-positive mES cells in a total of 200 mES cells passing quality filters. The x-axis shows EGFP signal intensity, and the y-axis shows counts of EGFP-positive mES cells.

Article Snippet: The PiggyBac transposase (PBase) effector vector (pCX-CAG>mPBase-PGK>HSV-TK) was constructed as follows: a custom fragment containing a Kozak sequence attached to the coding sequence (CDS) of mammalian codon-optimised PBase (mPBase) (GenBanK: EF587698.1 ) was synthesised by Integrated DNA Technologies (Coralville, IA).

Techniques: Plasmid Preparation, Transfection, Fluorescence, FACS, Concentration Assay