Navigation Links
MGH researchers develop faster method of engineering zinc-finger nucleases
Date:12/12/2010

A team led by Massachusetts General Hospital (MGH) researchers has developed a faster way to engineer synthetic enzymes that target specific DNA sequences for inactivation, repair or alteration. The report from the MGH Molecular Pathology Unit, being published online in Nature Methods, describes a highly effective but less labor-intensive way to generate powerful tools called zinc-finger nucleases (ZFNs).

"With our approach, called context-dependent assembly, any scientist can use either standard molecular biology techniques or commercial DNA synthesis to design ZFNs for their target gene of interest," says J. Keith Joung, MD, PhD, associate chief for Research in MGH Pathology, the study's senior author. "ZFNs are broadly applicable, powerful tools for manipulating the genomes of cells from various organisms including humans and may provide a way to efficiently correct gene mutations responsible for human disease, avoiding problems resulting from the imprecise nature of current gene therapy approaches using viral vectors."

Most human transcription factors that control whether a genetic signal is translated into a protein bind to specific DNA sequences using peptides called zinc fingers. Zinc-finger nucleases are synthetic "designer" proteins combining a zinc-finger domain, engineered to bind a particular DNA sequence, with an enzyme that breaks both DNA strands at the targeted site. While ZFNs have great potential, creating the customized proteins has been challenging.

In the simplest approach, called modular assembly, individual peptides are linked together like beads on a string to create a multi-finger protein theoretically able to recognize long DNA segments. Joung and others have shown that, in practice, modular assembly has a very low success rate for creating multi-finger proteins. This high failure rate is most likely due to "context-dependent" effects that individual zinc fingers can have on the DNA-binding activities of their neighboring fingers. Assembling peptides that don't work well together would be like trying to put together jigsaw puzzle pieces that don't fit.

In 2008, Joung and colleagues at the University of Minnesota and other institutions, members of the Zinc Finger Consortium, reported developing a method called OPEN (Oligomerized Pool ENgineering), which takes these context-dependent effects into account. But although OPEN works well, it can be labor intensive and extremely time consuming requiring up to a year for a lab to establish the technology and two months of work to generate desired ZFNs. To address these limitations, the MGH research team has assembled an extensive archive of zinc fingers known to work well when positioned together in essence puzzle pieces that already have been put together. Using this context-dependent method, the investigators were able to assemble dozens of ZFNs in as little as four days.

"With this archive in hand, any researcher can easily generate their own ZFNs in less than a week, and no special expertise is needed," Joung explains. "In addition to being much faster, context-dependent assembly can generate large numbers of ZFNs simultaneously, which is hard to do with OPEN because it is more labor intensive." As was the case with OPEN, the Joung lab and the Zinc Finger Consortium (http://www.zincfingers.org) will make the software and reagents required to practice context-dependent assembly available to all academic laboratories.

"One of the holy grails of genetics is the ability to make targeted changes to individual genes," says Laurie Tompkins, PhD, who over sees genetics grants at the National Institute of General Medical Sciences, one of the National Institutes of Health and a major supporter of this study. "Dr. Joung and his colleagues have developed an extraordinarily simple, efficient strategy for using zinc finger technology to swap out altered versions of genes for normal ones or vice versa providing basic scientists and clinicians alike with a broadly applicable research tool."

Adds Joung, an associate professor of Pathology at Harvard Medical School, "At this point, I believe that context-dependent assembly will have the biggest impact on researchers using ZFNs to genetically manipulate model organisms, possibly even models developed from pluripotent stem cells. Other big impacts should be enabling researchers to create knockout mutations in a large series of genes involved in a common pathway or related to a specific disease and to use ZFNs to create comprehensive collections of mutants for every gene in an organism." Joung is also a member of the MGH Center for Computational and Integrative Biology and Center for Cancer Research

The challenges posed to scientists interested in using ZFNs in their investigations were described in an article in the Fall 2010 issue of the MGH-sponsored magazine Proto, which can be accessed at http://protomag.com/assets/zinc-fingers-entry-fee.


'/>"/>

Contact: Sue McGreevey
smcgreevey@partners.org
617-724-2764
Massachusetts General Hospital
Source:Eurekalert

Related biology news :

1. Researchers discover how natural drug fights inflammation
2. Researchers devise computer model for projecting severity of flu season
3. NIH awards $6.4 million to Case Western Reserve School of Medicine researchers
4. Researchers discover a way to delay Christmas tree needle loss
5. Researchers: Include data about societal values in endangered species decisions
6. UT Southwestern researchers uncover culprits in life-threatening clotting disorder
7. SomaLogic researchers describe revolutionary new approach to protein analysis and application to early diagnosis of lung cancer
8. Researchers describe first functioning lipidome of mouse macrophage
9. Researchers find link between sugar, diabetes and aggression
10. Snakes on a rope: Researchers take a unique look at the climbing abilities of boa constrictors
11. Researchers kick-start ancient DNA
Post Your Comments:
*Name:
*Comment:
*Email:
(Date:6/27/2016)... Research and Markets has announced the addition ... to their offering. The report ... to grow at a CAGR of 12.28% during the period 2016-2020. ... in-depth market analysis with inputs from industry experts. The report covers ... The report also includes a discussion of the key vendors operating ...
(Date:6/22/2016)... , June 22, 2016  The American College of Medical ... Show Executive Magazine as one of the fastest-growing trade ... 25-27 at the Bellagio in Las Vegas ... highest percentage of growth in each of the following categories: ... companies and number of attendees. The 2015 ACMG Annual Meeting ...
(Date:6/22/2016)... 2016   Acuant , the leading ... has partnered with RightCrowd ® to ... Management, Self-Service Kiosks and Continuous Workforce Assurance. ... functional enhancements to existing physical access control ... with an automated ID verification and authentication ...
Breaking Biology News(10 mins):
(Date:6/27/2016)... PHILADELPHIA , June 27, 2016  Alex,s Lemonade ... announced that that it will open a state-of-the-art bioinformatics ... childhood cancer research. This announcement comes as Liz ... attends the National Cancer Moonshot Summit in ... Dr. Biden as a participant and advocate of pediatric ...
(Date:6/27/2016)... June 27, 2016  Global demand for enzymes ... through 2020 to $7.2 billion.  This market includes ... cleaning products, biofuel production, animal feed, and other ... and biocatalysts). Food and beverages will remain the ... increasing consumption of products containing enzymes in developing ...
(Date:6/27/2016)... , June 27, 2016 /PRNewswire/ - BIOREM Inc. (TSX-V: ... been advised by its major shareholders, Clean Technology Fund ... United States based venture capital funds ... of Biorem (on a fully diluted, as converted basis), ... disposition of their entire equity holdings in Biorem to ...
(Date:6/27/2016)... SAN DIEGO , June 27, 2016  Sequenom, ... company committed to enabling healthier lives through the development ... Supreme Court of the United States ... Federal courts that the claims of Sequenom,s U.S. Patent ... the patent eligibility criteria established by the Supreme Court,s ...
Breaking Biology Technology: