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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…. Sequence correction. xx. DRGFP reporter.

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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 Rad51… Sequence correction xx

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

  4. 041408 293T stable DRGFP (S8) pCMV-I-SceI no DNA 48 hrs 72 hrs

  5. Direct Repeat Reporters:recombination in cis

  6. 041408 293T DRGFP (cis) DRGFP+I-SceI I-SceI DRGFP

  7. 041408 293T DRGFP-lacO (cis) DRGFPlacO+I-SceI DRGFPlacO

  8. Exogenous Donor Reporter:recombination in trans

  9. 041408 293T ZFGFP + iGFP (trans) ZFGFP+iGFP ZFGFP+iGFP+I-SceI

  10. 041408 293T ZFGFP-lacO + iGFP (trans) ZFGFP-lacO ZFGFP-lacO+iGFP+I-SceI

  11. Too Much DNA?

  12. 050208 293T: Efficiency of Transient Transfection no DNA 62 ng eGFP 125 ng eGFP 250 ng eGFP 500 ng eGFP 1.0 g eGFP 2.0 g eGFP 4.0 g eGFP

  13. “Stable” DRGFP?

  14. “Stable” DRGFP (puroS) 48 hrs 73 hrs 98 hrs No DNA CMV-Sce

  15. “Stable” DRGFP (puroR) 48 hrs 73 hrs 98 hrs No DNA CMV-Sce

  16. Plans • Lines with integrated trans reporter • Enhance repair by tethering chromatin modifiers and repair factors • Back to B-cells

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