Research Idea Wiki Page

From OpenWetWare

(Difference between revisions)
Jump to: navigation, search
(Background Information)
(Background Information)
Line 11: Line 11:
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.
+
Works Cited
 +
Jantz, Derek, and Jeremy M. Berg. "Probing the DNA-Binding Affinity and Specificity of Designed Zinc Finger Proteins." Biophysical Journal 98.5 (2010): 852-60. Print.The study aims at evaluating factors that contribute to the accuracy and rapidness in the DNA binding of zinc finger nucleases (ZFN). the study uses a fluorescence anisotropy-based DNA binding assay to measure changes in specificity and affinity after implementing one residue substitution. The researches also varied external factors such as salt concentrations to see their effect.
 +
 
 +
Lederman, M. M., Adam Penn-Nicholson, Michael Cho, and Donald Mosier. "Biology of CCR5 and Its Role in HIV Infection and Treatment." JAMA: The Journal of the American Medical Association 296.7 (2006): 815-26. Print.This article describes the biological function and structure of the CCR5 receptor. It explains the process with which chemookine receptors are activated and transduce signals upon ligand binding. The CCR5 is particularly significant in HIV treatment because it has been identified as the point of entry for HIV in human cells.
 +
 
 +
Perez, Elena E., Jianbin Wang, Jeffrey C. Miller, Yann Jouvenot, Kenneth A. Kim, Olga Liu, Nathaniel Wang, Gary Lee, Victor V. Bartsevich, Ya-Li Lee, Dmitry Y. Guschin, Igor Rupniewski, Adam J. Waite, Carmine Carpenito, Richard G. Carroll, Jordan S Orange, Fyodor D. Urnov, Edward J. Rebar, Dale Ando, Philip D. Gregory, James L. Riley, Michael C. Holmes, and Carl H. June. "Establishment of HIV-1 Resistance in CD4+ T Cells by Genome Editing Using Zinc-finger Nucleases." Nature Biotechnology 26.7 (2008): 808-16. Print.This study analyzed the CCR5 delta 32 isoform as a potential model in HIV treatment using zinc finger nucleases (ZFN). This study developed a zinc finger nuclease with binding specificity to the CCR5 sequence, CCR5 ZFN. The ZFN disrupts the CCR5 function, analogous to the isoform present in HIV-immune individuals.
 +
 
 +
Urnov, Fyodor D., Jeffrey C. Miller, Ya-Li Lee, Christian M. Beausejour, Jeremy M. Rock, Sheldon Augustus, Andrew C. Jamieson, Matthew H. Porteus, Philip D. Gregory, and Michael C. Holmes. "Highly Efficient Endogenous Human Gene Correction Using Designed Zinc-finger Nucleases." Nature 435.7042 (2005): 646-51. Print.This study identifies the process with which zinc finger nucleases bind and modify target sequences of DNA. It evaluates the utility in using zinc finger nucleases in conjucntion with extrachromosomal DNA to insert sequences into the genome at target locations. The resulting data suggests T cells able to modified in high frequency suggesting ZFN as a tool to be used in immunotherapy.
===Purpose and Goals===
===Purpose and Goals===

Revision as of 11:30, 8 May 2013

Contents

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.

Works Cited Jantz, Derek, and Jeremy M. Berg. "Probing the DNA-Binding Affinity and Specificity of Designed Zinc Finger Proteins." Biophysical Journal 98.5 (2010): 852-60. Print.The study aims at evaluating factors that contribute to the accuracy and rapidness in the DNA binding of zinc finger nucleases (ZFN). the study uses a fluorescence anisotropy-based DNA binding assay to measure changes in specificity and affinity after implementing one residue substitution. The researches also varied external factors such as salt concentrations to see their effect.

Lederman, M. M., Adam Penn-Nicholson, Michael Cho, and Donald Mosier. "Biology of CCR5 and Its Role in HIV Infection and Treatment." JAMA: The Journal of the American Medical Association 296.7 (2006): 815-26. Print.This article describes the biological function and structure of the CCR5 receptor. It explains the process with which chemookine receptors are activated and transduce signals upon ligand binding. The CCR5 is particularly significant in HIV treatment because it has been identified as the point of entry for HIV in human cells.

Perez, Elena E., Jianbin Wang, Jeffrey C. Miller, Yann Jouvenot, Kenneth A. Kim, Olga Liu, Nathaniel Wang, Gary Lee, Victor V. Bartsevich, Ya-Li Lee, Dmitry Y. Guschin, Igor Rupniewski, Adam J. Waite, Carmine Carpenito, Richard G. Carroll, Jordan S Orange, Fyodor D. Urnov, Edward J. Rebar, Dale Ando, Philip D. Gregory, James L. Riley, Michael C. Holmes, and Carl H. June. "Establishment of HIV-1 Resistance in CD4+ T Cells by Genome Editing Using Zinc-finger Nucleases." Nature Biotechnology 26.7 (2008): 808-16. Print.This study analyzed the CCR5 delta 32 isoform as a potential model in HIV treatment using zinc finger nucleases (ZFN). This study developed a zinc finger nuclease with binding specificity to the CCR5 sequence, CCR5 ZFN. The ZFN disrupts the CCR5 function, analogous to the isoform present in HIV-immune individuals.

Urnov, Fyodor D., Jeffrey C. Miller, Ya-Li Lee, Christian M. Beausejour, Jeremy M. Rock, Sheldon Augustus, Andrew C. Jamieson, Matthew H. Porteus, Philip D. Gregory, and Michael C. Holmes. "Highly Efficient Endogenous Human Gene Correction Using Designed Zinc-finger Nucleases." Nature 435.7042 (2005): 646-51. Print.This study identifies the process with which zinc finger nucleases bind and modify target sequences of DNA. It evaluates the utility in using zinc finger nucleases in conjucntion with extrachromosomal DNA to insert sequences into the genome at target locations. The resulting data suggests T cells able to modified in high frequency suggesting ZFN as a tool to be used in immunotherapy.

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.

Personal tools