1 / 8

Optimizing efficiency and safety of targeted gene correction

Optimizing efficiency and safety of targeted gene correction. NHEJ. Cleave or nick. religation in situ. Homology-directed repair. 25 20 15 10 5 0. 21-fold. Foci per nucleus. 0.4 21 1. - H2AX foci induced by cleavase but not nickase. Merge/DAPI. - H2AX. -.

addo
Download Presentation

Optimizing efficiency and safety of targeted gene correction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Optimizing efficiency and safety of targeted gene correction NHEJ Cleave or nick religation in situ Homology-directed repair

  2. 25 20 15 10 5 0 21-fold Foci per nucleus 0.4 21 1 -H2AX foci induced by cleavase but not nickase Merge/DAPI -H2AX - -H2AX: cleavase/nickase I-AniI Cleavase I-AniI Nickase

  3. A non-homologous end joining (NHEJ) reporter LHE LHE P CD8+, H2Kd-, CD4- H2-Kd CD8 CD4 polyA LHE LHE P polyA H2Kd+, CD4- H2-Kd CD4 CD8 P H2Kd-, CD4+ CD4 LHE P polyA H2Kd-, CD4- CD8 2-Kd CD4 Reporter: Cleavage-associated genomic instability Products: Inversion following cleavage at both sites Deletion following cleavage at both sites Processing and NHEJ of a single cleavage event Guirouilh-Barbat et.al. 2004

  4. Gene correction and NHEJ:a model for optimization gfp- - - - NHEJ

  5. Gene correction and NHEJ:a model for optimization gfp- - - - NHEJ transfect donor template LHE GFP

  6. Gene correction and NHEJ:a model for optimization GFP+ - x - - NHEJ

  7. Correction @ A  Mutation @ B no I-SceI H2-Kd stained + I-SceI stained H2-Kd + I-SceI unstained 0.42% 0.37% 0.004% GFP 0.29% 0.003% 0.23% 0.11% 14.5% 84.4% 2.4% 97.3% H2-Kd (PE)

  8. Correction @ A  Mutation @ B no I-SceI H2-Kd stained + I-SceI stained H2-Kd + I-SceI unstained 1.96% 1.62% 0.001% GFP 1.34% 0% 0.77% 0.67% 10.9% 86.6% 9.7% 89.4% H2-Kd (PE)

More Related