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Homology-directed gene repair

Homology-directed gene repair. exogenous repair substrate. I-SceI. xx. sister chromatids. xx. Rad51…. Sequence correction. xx. Homology-directed gene repair. exogenous repair substrate. I-SceI. xx. sister chromatids. xx. Rad51 K313Q. Sequence correction. xx. BcgI.

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Homology-directed gene repair

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  1. Homology-directed gene repair exogenous repair substrate I-SceI xx sister chromatids xx Rad51… Sequence correction xx

  2. Homology-directed gene repair exogenous repair substrate I-SceI xx sister chromatids xx Rad51K313Q Sequence correction xx

  3. BcgI GFP reporter w/ exogenous donor exogenous repair substrate CA puroR I-SceI STOP gene conversion CA GFP+ puroR BcgI

  4. DRGFP reporter CA puroR I-SceI STOP BcgI gene conversion CA GFP+ puroR BcgI BcgI

  5. 013108 Nalm6 Transfection no DNA 24hr 1µg pmaxGFP 24hr

  6. Nalm-6 DRGFP • Functional screen of puroR cell lines • in 1?/24 I-SceI  nuc dependent GFP+

  7. 030308 Nalm-6 DRGFP#13 72 hrs No I-SceI 72 hrs + I-SceI No TSA + TSA

  8. Possibilities • Check more clones • Chromatin • acetylation • methylation • Nalm-6 defect • Msh2-… • try other cell lines

  9. David Bednarski Amber Caracol Jason Cummings Luther Davis Johanna Eddy Nicolle Hamilton Jana Mandic Ellen Ordinario Liz Sacho Michael Stevenson Aarthy Vallur Munehisa Yabuki Nancy Maizels

  10. GFP x FL2-H No DNA + pEGFP

  11. New GFP reporter w/ exogenous donor exogenous repair substrate CMV lacO NeoR intron I-SceI… STOP (x2)

  12. 4 4 10 4 10 10 0 0 0 3 3 10 3 10 10 FL1-H: GFP FL1-H: GFP FL1-H: GFP 2 2 10 2 10 10 1 1 10 1 10 10 0 0 10 0 10 10 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 FL2-H: FL2-Height FL2-H: FL2-Height FL2-H: FL2-Height 4 4 4 10 10 10 13.3 19.5 29.3 3 3 3 10 10 10 FL1-H: GFP FL1-H: GFP FL1-H: GFP 2 2 2 10 10 10 1 1 1 10 10 10 0 0 0 10 10 10 0 1 2 3 4 0 1 2 3 4 0 1 2 3 4 10 10 10 10 10 10 10 10 10 10 10 10 10 10 10 FL2-H: FL2-Height FL2-H: FL2-Height FL2-H: FL2-Height Transfection Efficiency 72 hrs 96 hrs 48 hrs No pGFP + pGFP

  13. 1000 1000 1000 800 800 800 600 600 600 SSC-H: Side Scatter SSC-H: Side Scatter SSC-H: Side Scatter 83.1 83.2 76.3 400 400 400 0.81 1.23 3.19 200 200 200 0 0 0 0 200 400 600 800 1000 0 200 400 600 800 1000 0 200 400 600 800 1000 FSC-H: Forward Scatter FSC-H: Forward Scatter FSC-H: Forward Scatter I-SceI Expression  Dead Cells? No DNA pEGFP pCMV-Sce 48 hrs 030308

  14. Nalm-6 DRGFP

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