View - ResearchGate

Engineering Cys2His2 Zinc Finger Domains 33310. pAC-αGal4 encodes a fusion protein consisting of the N-terminal domain andinterdomain linker of the E. coli RNA polymerase α-subunit, fused to amino acidresidues 58–97 of the yeast Gal4 protein. Expression of the αGal4 hybrid proteinfrom pAC-αGal4 is directed by a strong, IPTG-inducible semisyntheticlpp/lacUV5 promoter. The pAC-aGal4 plasmid possesses a p15A origin of replicationand confers resistance to chloramphenicol.11. Typically, it is aimed for a threefold oversampling of the size of the randomizedlibrary being interrogated and for a fivefold ratio of total cells to transformedcells. For example, for a randomized library with a complexity of approx 2 × 10 8 ,one would aim to plate a total of approx 6 × 10 8 transformed cells and of morethan 3 × 10 9 total cells on the selection plate.12. It has been found that these triple washes are critical for obtaining good qualitysequencing reads. It is believed that these washes help reduce contaminatingendonuclease activity from the endA + selection strains.13. 0.1X EB is buffer EB from the QIAgen miniprep kit diluted 10-fold with ddH 2O.AcknowledgmentsThis work was supported by grants from the National Institutes of Health (K08DK002883 and R01 GM069906) and start-up funds from the MassachusettsGeneral Hospital Department of Pathology. J.K.J. dedicates this work to Robert L.Burghoff, Ph.D., who always taught and shared his best protocols.References1. Jamieson, A. C., Miller, J. C., and Pabo, C. O. (2003) Drug discovery with engineeredzinc-finger proteins. Nat. Rev. Drug Discov. 2, 361–368.2. Beerli, R. R. and Barbas, C. F., 3rd. (2002) Engineering polydactyl zinc-fingertranscription factors. Nat. Biotechnol. 20, 135–341.3. Blancafort, P., Segal, D. J., and Barbas, C. F., 3rd. (2004) Designing transcriptionfactor architectures for drug discovery. Mol. Pharmacol. 66, 1361–1371.4. Durai, S., Mani, M., Kandavelou, K., Wu, J., Porteus, M. H., and Chandrasegaran, S.(2005) Zinc finger nucleases: custom-designed molecular scissors for genomeengineering of plant and mammalian cells. Nucleic Acids Res. 33, 5978–5990.5. Porteus, M. H. (2005) Mammalian gene targeting with designed zinc fingernucleases. Mol. Ther. 13(2), 438–446.6. Porteus, M. H. and Baltimore, D. (2003) Chimeric nucleases stimulate genetargeting in human cells. Science 300, 763.7. Porteus, M. H. and Carroll, D. (2005) Gene targeting using zinc finger nucleases.Nat. Biotechnol. 23, 967–973.8. Urnov, F. D., Miller, J. C., Lee, Y. L., et al. (2005) Highly efficient endogenoushuman gene correction using designed zinc-finger nucleases. Nature 435, 646–651.9. Alwin, S., Gere, M. B., Guhl, E., et al. (2005) Custom zinc-finger nucleases for usein human cells. Mol. Ther. 12(4), 610–617.10. Choo, Y. and Klug, A. (1995) Designing DNA-binding proteins on the surface offilamentous phage. Curr. Opin. Biotechnol. 6, 431–436.