Research Idea Wiki Page: Difference between revisions
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Zinc Finger nucleases (ZFN) are tools that can be used to modify the human genome in a very target specific manner. The nucleases consist of two domains: DNA-binding domain, which locates and binds the target sequence in the genome, and the nuclease domain, that cleaves and modifies the target sequence using homologous recombination with extrachromosomal DNA (Urnov et al. 2005). Homology-directed repair is a mechanism typically used to repair damaged DNA by introducing new modified DNA sequences into the genome at target locations. | Zinc Finger nucleases (ZFN) are tools that can be used to modify the human genome in a very target specific manner. The nucleases consist of two domains: DNA-binding domain, which locates and binds the target sequence in the genome, and the nuclease domain, that cleaves and modifies the target sequence using homologous recombination with extrachromosomal DNA (Urnov et al. 2005). Homology-directed repair is a mechanism typically used to repair damaged DNA by introducing new modified DNA sequences into the genome at target locations. | ||
The high specificity with which zinc finger nucleases function does not directly translate to a high affinity for the targeted sequence (Jantz and Berg 2010). We propose to modify the commonly used CCR5-ZFN model developed by Perez et al in 2008 using site-directed mutagensis to increase binding affinity of the nuclease to the CCR5 sequence. To measure changes in binding affinity, we could compare the levels of CCR5 expression using a fluorescence based anisotropy DNA-binding assay as described by Jantz and Berg to measure nuclease binding affinity. | The high specificity with which zinc finger nucleases function does not directly translate to a high affinity for the targeted sequence (Jantz and Berg 2010). We propose to modify the commonly used CCR5-ZFN model developed by Perez et al in 2008 using site-directed mutagensis to increase binding affinity of the nuclease to the CCR5 sequence. To measure changes in binding affinity, we could compare the levels of CCR5 expression using a fluorescence based anisotropy DNA-binding assay as described by Jantz and Berg to measure nuclease binding affinity. | ||
References. | |||
===Purpose and Goals=== | ===Purpose and Goals=== | ||
Revision as of 09:04, 8 May 2013
Project Overview
We are planning to use site-directed mutagenesis to increase the binding affinity of CCR5 zinc finger nucleases. This mutation aims to increase the level CCR5 receptor disruption in genetically haploid cells in an effort to facilitate HIV therapy.
Background Information
CCR5 is a chemokine receptor located on the membranes of T cells that has been identified to be the source of HIV entry in human cells. CCR5 delta 32 is an isoform of CCR5 found in certain Northern European individuals that confers resistance, or immunity, to HIV. This deletion is known to have minimal consequences due to the overlapping relationships between different ligands and chemokine receptors (Lederman et al 2005) Zinc Finger nucleases (ZFN) are tools that can be used to modify the human genome in a very target specific manner. The nucleases consist of two domains: DNA-binding domain, which locates and binds the target sequence in the genome, and the nuclease domain, that cleaves and modifies the target sequence using homologous recombination with extrachromosomal DNA (Urnov et al. 2005). Homology-directed repair is a mechanism typically used to repair damaged DNA by introducing new modified DNA sequences into the genome at target locations. The high specificity with which zinc finger nucleases function does not directly translate to a high affinity for the targeted sequence (Jantz and Berg 2010). We propose to modify the commonly used CCR5-ZFN model developed by Perez et al in 2008 using site-directed mutagensis to increase binding affinity of the nuclease to the CCR5 sequence. To measure changes in binding affinity, we could compare the levels of CCR5 expression using a fluorescence based anisotropy DNA-binding assay as described by Jantz and Berg to measure nuclease binding affinity.
References.
Purpose and Goals
Project Details
Predicted Outcomes
Needed Resources
Zinc Finger nucleases specific to CCR5. CCR5 delta 32 sequence and a construct with the CCR5delta32 sequence within arms of CCR5 sequences. KBM7 cells that are haploid for every chromosome except for chromosome 8. It will be easier to see the effects of a change in haploid cells because there won't be an ambiguous heterozygous phenotype.
