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Matthews Lecture 2_final 140115

Clustered regulatory interspaced short palindromic

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Unformatted text preview: pol II CYP3A4 Phase II MDR XREM Changes in gene expression XREM- xenobio/c- responsive enhancer module 47 Response Elements in CYP3A PXR is an key regulator of CYP3A family XREM- xenobio/c- responsive enhancer module 48 Response Elements Recognized by PXR/RXR Kliewer, S. A. et al. Endocr Rev 2002;23:687-702 49 Copyright ©2002 The Endocrine Society AGGTCAnnnAGGTCA AGGTCAnnnnAGGTCA TGACCTnnnnnnAGGTCA AGGTCA Pregnane X receptor (PXR), NR1I2 Human not rodent rodent not human (St. John’s Wort) 51 PCN = 5- pregnen- 3beta- ol- 20- one- 16alpha- carbonitrile Transient transfec/on Receptor- reporter gene (luciferase) assays and compe//ve binding assays PXR has a large binding pocket that can accommodate a number of structurally diverse ligands Gene knockout in mice Gene dele/on - Knockout mice Humanized mouse models for PXR Gene knock- in mPXR hPXR Knockout or knock-in Cell lines Non-murine models ZFN Pair recognizes and heterodimerizes around target site ZFN Pair makes dsDNA break and dissociates from DNA NO REPAIR TEMPLATE Co-transfection with ZFN Pair 1-20% resulting in gene deletion using nonhomologous end joining REPAIR TEMPLATE Co-transfection with ZFN Pair 1-20% contain Gene Integration in Target site using homologous 56 recombination Zinc finger nuclease (ZFN) ZFNs consist of two func?onal domains: • DNA- binding domain comprised of a chain of zinc finger proteins • DNA cleaving domain comprised of the nuclease domain of FokI (restric/on endonuclease) • FokI must dimerize to make a double- stranded DNA break • a pair of ZFN are required to cleave double- stranded DNA 57 Zinc Finger Protein Design Using Twofinger Modules (6 bp subsites) Four-finger ZFP (12 bp target site) Build 4, 5, or 6-finger proteins that target 12, 15, or 18bp respectively ZFN sites are spaced by 5-7 bp 58 Alternative and strategies •  transcrip/on ac/vator- like effectors (TALENs) •  Recognize single nucleo/des •  Increase flexibility compared with ZFNs •  clustered regulatory interspaced short palindromic repeats (CRISPRs) •  •  Derived from acquired immunity...
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