NEWS OF THE WEEK
IDENTIFYING MODIFIED CELLS MOLECULAR BIOLOGY: Technique tags and enriches cells genetically altered by nucleases
MODIFIED WITH PERMISSION FROM NAT. METHODS
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NEW TECHNIQUE identifies and enriches cells
whose genomes have been modified by customdesigned nucleases. The method could promote the use of nucleases to tweak genomes for various applications— for example, to study the roles of specific genes and to develop gene therapies. Zinc finger nucleases and transcription-activator-like effector nucleases customized to target specific genes are available commercially and can also be designed by researchers in their laboratories. They bind and cleave specific gene sequences in cells, causing the genes to be disabled or modified. A key challenge in using nucleases is that some modify only a small fraction of cells, making it difficult to run experiments. Previous methods to enrich altered cells are used only infrequently. Now, a South Korean team has developed a simple and convenient method to tag nuclease-modified cells fluorescently, making it possible to enrich them up to 92-fold (Nat. Methods, DOI: 10.1038/nmeth.1733). The team was led by assistant professor of biomedical science and engineering Hyongbum Kim of Hanyang University, in Seoul, and associate professor of chemistry Jin-Soo Kim of Seoul National University. The technique involves adding circular DNAs known as plasmids to cells that are to be modified by
nucleases. The plasmids contain a copy of the gene targeted by the nucleases and genes for a red and green fluorescent fusion protein. If the targeted sequence is modified by nucleases, the fluorescent fusion protein is expressed. Cells exhibiting the glow from that plasmid protein can then be recognized and separated by flow cytometry. The glow appears only when the target gene is modified in the plasmid, not in the genome. But the target gene is changed at the same time in both locations frequently enough that the technique works well, enabling genomically altered cells to be enriched substantially, albeit not completely. Plasmid
Flow cytometry
Genome
“The technique is clever and should be broadly applicable for modifying genes in cultured cells,” comments John H. Wilson, a specialist in gene therapy at Baylor College of Medicine. “It is quick and easy and could be used by anyone. Perhaps it might allow less efficient nucleases to become more useful or might prove beneficial in cells in which even ‘good’ nucleases don’t work well.” Carlos F. Barbas III of Scripps Research Institute, a specialist in zinc finger nucleases, notes that the marker technique is “traceless.” Other enrichment methods “introduce another gene that needs to be subsequently removed from cells, so those approaches leave their footprints in the genome, whereas this one does not,” he says. “It has some real advantages, and I think many labs will use it.” The customized reporter plasmid technology is available commercially from ToolGen, in Seoul, a company cofounded by Jin-Soo Kim.—STU BORMAN
Nucleases modify (black band) a target genomic sequence in only a fraction of cells (left). Plasmid expression of red and green fluorescent fusion protein occurs mostly in cells with nucleasemodified genomes. Flow cytometry can detect the fluorescence and enrich the cells (right).
BIOPHARMA Regulatory pressures trigger a drop in venture capital investments, report finds A report by the National Venture Capital Association (NVCA) cites regulatory challenges, specifically from the Food & Drug Administration, as the most significant factor driving venture capital away from start-up biopharmaceutical and medical device companies in the U.S. According to “Vital Signs: The Threat to Investment in U.S. Medical Innovation and the Imperative of FDA Reform,” U.S. venture capital firms are decreasing their first-time investments in companies in the sector, cutting back on commitments to research in prevalent disease areas, and
shifting their focus to Europe and Asia. Of the approximately 150 investment firms interviewed for the report, 39% have reduced their first-time investments in U.S.-based life sciences companies over the past three years, and the same percentage plans to decrease investment over the next three years. The drop-off comes as overall investment in the life sciences is rising. According to NVCA, total dollar investment in the sector in the U.S. jumped 37% in the second quarter of 2011, but that increase represents follow-on investments in cur-
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rent projects, which are generally higher than initial investments. “As a driver of our global economy, the FDA should constantly examine its regulatory framework with a 21st-century lens,” Sen. Michael F. Bennet (D-Colo.) said at a press conference about the report on Capitol Hill earlier this month. FDA recently outlined steps it can take to spur biotech and pharmaceutical innovation. The report details outreach services to support small firms in product development and expedite review processes (C&EN, Oct. 10, page 31).—RICK MULLIN
