Titlebook: Engineered Zinc Finger Proteins; Methods and Protocol Joel P. Mackay,David J. Segal Book 2010 Springer Science + Business Media, LLC 2010 A

无能的人

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https://doi.org/10.1007/978-3-662-63453-0re the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.

祸害隐伏

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Quantification of Zinc Finger Nuclease-Associated Toxicityre the nuclease is properly bound to the DNA. Here, we describe two different methods to quantify ZFN-associated toxicity: the genotoxicity assay is based on quantification of DSB repair foci induced by ZFNs whereas the cytotoxicity is based on assessing cell survival after application of ZFNs.

Conquest

Generation of Cell-Permeable Artificial Zinc Finger Protein Variantsts by using plasmids or viral vectors. As an alternative delivery method of ZFP variants, we developed cell-permeable ZFP variants by fusing cell-penetrating peptides to ZFP variants. We will describe how to generate cell-permeable artificial ZFP variants and how to examine the cell permeabilities by immunofluorescent staining.

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Modulation of Gene Expression Using Zinc Finger-Based Artificial Transcription Factorsion molecule and against the promoter of the RNA component of telomerase. Methods to assess DNA binding of the engineered ZFP as well as to determine and improve the cellular effect of ATFs on (endogenous) promoter activity are described.

Cytology

In Vitro Assessment of Zinc Finger Nuclease Activityuding the specificity of DNA binding, the kinetics of dimerization of the two ZFN subunits, and the catalytic activity. In order to investigate these parameters individually, a cell-free system that models these reactions is essential. Here, we present a simple and fast method for the functional testing of ZFNs in vitro.

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Engineered Zinc Finger Proteins for Manipulation of the Human Mitochondrial Genomein a sequence-specific manner. Here, basic methods that are helpful in adapting the engineered zinc finger technology for targeting the DNA in mitochondria are presented with the main emphasis on mitochondrial import of zinc finger proteins.

Glycogen

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Silencing of Gene Expression by Targeted DNA Methylation: Concepts and Approachestable repression of known target genes. We review the literature on targeted DNA methylation and gene silencing, summarize the achievements and the challenges that remain, and discuss technical issues critical for this approach.