Worcester, MA, USA. A new technique that improves the ability of scientists to target individual genes for inactivation has broad potential implications for basic science research and treatment. Two scientific teams working with zebrafish — commonly used as a model organism in biomedical research — to develop a method to create and deliver a tailor-made “restriction enzyme” that inactivates a specific gene in the embryo.

 

“The best way to figure out what a gene does in an organism is to replace it with a non-functional version, breed the individual, and then look at the offspring to see what’s wrong with them,” said Laurie Tompkins, Ph.D., who oversees genetic mechanisms grants at the National Institute of General Medical Sciences.

 

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Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Xiangdong Meng, Marcus B Noyes, Lihua J Zhu, Nathan D Lawson & Scot A Wolfe. Nature Biotechnology. Online: 25 May 2008. doi: 10.1038 / nbt1398

Tompkins says “The problem is that it’s hard to swap in non-functional genes that are inherited by the offspring. These investigators have devised a way to do this, which will enable many scientists to answer questions that were previously out of reach.”

 

“We believe that this work will fundamentally change how researchers make knockouts — research organisms in which one or more genes have been genetically engineered to be turned off — in many model organisms,” said Dr. Wolfe. “In this paper, we demonstrate the feasibility of this approach for gene inactivation using the zebrafish, but we believe that this technology should be applicable to other vertebrate and non-vertebrate systems with exciting implications for the development of new models for the study of human disease.”

 

“The zebrafish has really become quite established as a model organism in the past several years,” said Dr. Lawson. “I began using the zebrafish model to study angiogenesis because of its external development and transparent embryos — we can actually watch blood vessels as they grow in the zebrafish embryo. This allows us to gain novel insights into this process that are not easy to make in mouse models.

 

However, we had not previously been able to directly knock out a gene of interest, an approach available in the mouse. The work we have done with the Wolfe lab will open up completely new avenues for our own research and will further strengthen the use of the zebrafish model. More significantly, this technique will now allow us to make zebrafish models that may provide insight into the progression of human vascular disease.”

 

This research was supported by grants from the National Heart, Lung and Blood Institute and the National Institute of General Medical Sciences.

 

Targeted gene inactivation in zebrafish using engineered zinc-finger nucleases. Xiangdong Meng, Marcus B Noyes, Lihua J Zhu, Nathan D Lawson & Scot A Wolfe. Nature Biotechnology. Online: 25 May 2008. doi: 10.1038 / nbt1398

Abstract. Direct genomic manipulation at a specific locus is still not feasible in most vertebrate model organisms. In vertebrate cell lines, genomic lesions at a specific site have been introduced using zinc-finger nucleases (ZFNs). Here we adapt this technology to create targeted mutations in the zebrafish germ line. ZFNs were engineered that recognize sequences in the zebrafish ortholog of the vascular endothelial growth factor-2 receptor, kdr (also known as kdra). Co-injection of mRNAs encoding these ZFNs into one-cell-stage zebrafish embryos led to mutagenic lesions at the target site that were transmitted through the germ line with high frequency. The use of engineered ZFNs to introduce heritable mutations into a genome obviates the need for embryonic stem cell lines and should be applicable to most animal species for which early-stage embryos are easily accessible.

 

 

 

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