This photo was created using Mextures app, which is a very powerful and unique photo editor with the ability to stack many different layers and blending modes. If you want to follow along, you can download a full resolution version of this photo by clicking on it and then selecting Download at the bottom of the image popup window. The determination of complete genome sequences for a wide variety of experimental organisms established a first step in the search for an in-depth understanding of the complex genetic functions that define living entities. Starting with Nobel Prize winner Mario Capecchi’s earliest work, it was discovered that the HR process could be harnessed and directed in mouse embryonic stem cells (MESCs) by introducing exogenous DNA as a repair template to induce precise point mutations, gene deletions, and gene insertions.
In the early 1990s, the discovery and manipulation of zinc finger protein domains started a field of research that enabled a very targeted approach to genetic manipulation in higher level eukaryotes that was one thousand times more efficient than the prevailing HR-based mechanism. More important was the discovery that the DNA-binding properties of zinc fingers could be fused to proteins with DNAcutting function.
The power of a ZFN-induced double-strand break (DSB) comes from the fact that a DSB can be repaired by one of two repair processes: non-homologous end joining (NHEJ) or homologydependent repair (HDR). ZFN-mediated gene targeting is a very powerful and versatile tool for targeted genome editing of a wide range of organisms and cells. Targeted genome editing with ZFNs has a wide variety of applications, and three key modes of action have been a focus thus far: targeted gene knockouts, targeted gene integration, and targeted gene correction.
ZFNs offer a rapid single-step approach to targeted gene knockout in mammalian cells, using NHEJ.
ZFN-mediated gene integration allows users to place exogenous genetic sequences precisely into targeted sites of the genome.
In order to gain acceptance as a robust methodology, any means of targeted gene integration should occur at a frequency high enough to obviate the need for time-consuming selection of cells carrying the desired gene.
Efficient correction of deleterious changes in genes would provide researchers with the ability to fully understand the impact of the mutation on the organism, improve cell lines that are valuable research tools or have commercial importance, and of course correct genetic defects that can cause human disease. Since X-linked severe combined immunodeficiency disease (SCID) has been the target of gene therapy efforts, it was used as a model system to demonstrate the utility of ZFN-mediated gene correction.8 SCID is caused by mutations in the IL2R gene. Successful genetic manipulation of an organism requires that progenitor and stem cells be modified efficiently ex vivo by gene transfer, with subsequent culturing and re-infusion into the organism to establish a stable and heritable change. Sangamo BioSciences has been a major leader in the development of zinc finger protein technologies as a means of genespecific editing.
In 2007, Sigma-Aldrich formed an exclusive partnership with Sangamo to make ZFN technology easily available to research scientists around the world through its CompoZr™ brand line of products and services.
The CompoZr Custom ZFN offering, launched in Fall 2008, supplies partners with Zinc Finger Nucleases that have been validated to target and edit customer-defined genes of interest.
Future CompoZr products based on ZFN technology, available in 2009, will include ZFNs targeting popular genes, gene families, and pathways. The ability to routinely edit genes in cell lines, animals and plants has long been a goal sought after by researchers seeking an in-depth understanding of complex genetic functions and applying that knowledge to develop new pharmaceuticals, improve agricultural productivity, and even cure human disease through gene therapy. After configuring your server as described in Configuration for WebDAV, users who meet the client-side requirements listed in the Requirements topic may start editing document attachments or documents using the File field (e.g. You have learned how document attachments or documents using the File field can be edited using a client application when WebDAV integration is enabled.

Note: The authors of the following samples have provided LetPub with their permission to disclose their manuscripts. I particularly like how I was able to emphasize the warm evening light at the top part of the image while leaving the bottom in the shadow and relatively cold.
Extensive genetic, biochemical, cytological and physiological analyses are now required to correlate genome sequence with the next level of understanding of genetic function.
The most well studied methods have relied on the process of homologous recombination (HR), a naturally occurring DNA-repair mechanism found in most cells that uses the second copy of a chromosome as a template to repair damage to a gene. This approach to gene targeting led to the development of knockout mice as a research tool, still considered the gold standard for many functional genomics studies.
Zinc fingers are structures of about 30 amino acids held together by a zinc ion, and they determine the binding specificity of the most abundant class of transcription factors found in a wide range of species. For instance, four zinc finger units can be assembled into a zinc finger chain that recognizes a 12 base pair DNA sequence. Each Zinc Finger Nuclease (ZFN) consists of two functional domains: a) A DNA-binding domain comprised of a chain of two-finger modules, each recognizing a unique hexamer (6 bp) sequence of DNA. This can result in as much as 20% of the cells containing a gene insertion at the target site (Figure 2). ZFN-mediated genome editing takes place in the nucleus when a pair of ZFNs targeting the user’s gene of interest (GOI) is delivered transiently into a cell line, either by transfection or electroporation. The process of ZFN-mediated gene modification is as simple as a single transfection experiment, and edits occur in as little as three days.
As an example, ZFNs have been used to target the dihydrofolate reductase (DHFR) gene in a Chinese hamster ovary (CHO) cell line diploid for a functional DHFR generated gene. Up to 32% of embryos injected with ZFNs targeting golden carried clones of unpigmented cells in the eye, representing loss of golden gene function.
This newfound ability will not only lead to a new generation of functional genomics studies, but will have great impact in the drug discovery world as it will the allow creation of new cell lines containing fusion tags or reporters that can be used for more efficient cell-based screening. ZFN-mediated gene correction provides this capability across a wide range of organisms and cell types. Using ZFNs, targeted alterations were achieved in this gene in more than 18% of the recipient cells – without selection. This is of course a requirement for human gene therapy, and again SCID has been used as a model system to demonstrate the effectiveness of ZFN-mediated correction. Currently, Sangamo is focused on using these technologies to develop gene regulation and correction therapeutics. Deliverables of the CompoZr Custom ZFN service include ready-to-transfect amounts of Zinc Finger Nuclease, as well as controls and reagents for screening for mutational events. Zinc finger nuclease technology promises to realize this goal by providing the tools to precisely and efficiently induce genetic changes in systems ranging from human stem cell lines to fruit flies, plants and a host of other organisms. The same Edit in client application () icon is displayed on the Form tab of documents using the File field (e.g. Clicking the icon opens the document in a client application associated with the particular file extension (e.g.

Now close the client application and try to open the document again, either from the UI or from the live site.
You should see that the file that you modified has been saved to the server and the version that you have just opened contains the modifications you made to it in the previous step. As such, technologies that allow researchers to routinely and efficiently edit the genomes of virtually any species, by directing mutations in a truly targeted fashion, would greatly enhance the understanding of basic biology, and potentially lead to novel ways of treating human disease. However, HR was found to be woefully inefficient in many other relevant model systems, including mammalian somatic cells, in which the rate of HR is much lower than the rate of HR in MESCs. This chain can then be fused to the catalytic domain of the endonuclease FokI to create a zinc finger nuclease (ZFN). With as much as 20% of the cells undergoing targeted genomic editing, selection markers are most often unnecessary. CHO cells were chosen for this experiment as they are often the system of choice for the production of therapeutic recombinant proteins.
Injecting the ZFN targeting the ntl gene resulted in 27% of the embryos showing the stunted tail growth phenotype. Using a gene delivery approach based on integrase-defective lentiviral vectors, ZFNs have been used to generate permanent and heritable modifications of the IL2R gene in hematopoietic progenitor cells as well as human embryonic stem cell lines at high frequencies, allowing rapid, selection-free isolation of clonogenic cells with the desired genetic modification.9 Up to 50% targeted gene addition was achieved in a panel of recipient human cell lines, and up to 5% of human embryonic stem cells. They are currently conducting phase 1 and 2 trials for application of the up-regulation of endogenous VEGF-A in the treatment of diabetic neuropathy.
Sangamo BioSciences has led the way in developing this revolutionary technology by designing ZFNs that are exquisitely specific and efficient, and demonstrating their utility for generating targeted gene knockouts, gene integration and even gene correction in relevant biological systems.
Microsoft Office Excel, PowerPoint, Paint, etc.) can be performed exactly the same way - all you need to do is click the Edit in client application () icon, edit the file and save it in the client application.
With the protospacer-adjacent motif (PAM - the sequence NGG) present at the 3? end, Cas9 will unwind the DNA duplex and cleave both strands upon recognition of a target sequence by the guide RNA.
For this reason HR never found its footing as a robust means of mutatgenesis in cell types commonly used in functional genomics, target validation, and protein production settings. Once high-quality ZFNs have been constructed, isogenic stable cell lines containing the modifications can be generated in as little as a month through simple dilution cloning.
With alternative knockout methodologies such as antisense and RNAi, possible toxicity effects of long-term expression of these polynucleotides, as well as the incomplete phenotypic penetrance that may arise from partial knockdown using these methods, may prove undesirable.
ZFN-mediated gene correction can clearly provide highly efficient gene correction that can be used in a wide variety of organisms. Induction and repair of zinc-finger nuclease-targeted double-strand breaks in caenorhabditis elegans somatic cells. Targeted gene addition into a specified location in the human genome using designed zinc finger nucleases. Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery.

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