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

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

3. DRGFP reporter puro R I-SceISTOPBcgI puro R BcgI GFP + gene conversion CACA CACA

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

5. Direct Repeat Reporters: recombination in cis

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

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

8. Exogenous Donor Reporter: recombination in trans

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

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

11. Too Much DNA?

12. 293T: Efficiency of Transient Transfection 050208 500 ng eGFP no DNA 1.0  g eGFP2.0  g eGFP4.0  g eGFP 62 ng eGFP125 ng eGFP250 ng eGFP

13. “Stable” DRGFP?

14. “Stable” DRGFP (puro S ) No DNA CMV-Sce 48 hrs73 hrs98 hrs

15. “Stable” DRGFP (puro R ) No DNA CMV-Sce 48 hrs73 hrs98 hrs

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