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. Author manuscript; available in PMC: 2022 Jun 9.
Published in final edited form as: Nat Biotechnol. 2021 Dec 9;40(5):731–740. doi: 10.1038/s41587-021-01133-w

Extended Data Fig. 8. TwinPE and Bxb1-mediated inversion in HEK293T GFP reporter cells.

Extended Data Fig. 8

(a) The lentiviral fluorescent reporter construct used to assess inversion efficiency with twinPE and Bxb1 recombinase. The reporter contains an EF1α promoter followed by an inverted H2B-EGFP coding sequence that is flanked by partial AAVS1 DNA sequence, an internal ribosome entry site (IRES), and a puromycin resistance gene. Successful installation of opposite-facing attB (left) and attP (right) sequences at the AAVS1 target sequences and subsequent inversion by Bxb1 corrects the orientation of GFP for functional expression. (b) The fluorescent reporter construct was stably integrated into HEK293T cells via lentiviral transduction and puromycin selection. The polyclonal GFP reporter cell line was then transfected with twinPE plasmid components (PE2 and four pegRNAs) and varying amounts of Bxb1 plasmid for single-transfection inversion. Cells were analyzed by flow cytometry and gated for live single cells. Quantification of GFP positive cells by flow cytometry. Values and error bars reflect the mean of two independent biological replicates.