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TSRI chemists unveil new technique for marking and selecting cells
LA JOLLA, Calif.—Seeking an easier and cheaper way to mark cells for selectability in molecular biology research, chemists at The Scripps Research Institute (TSRI) have designed a new method that allows scientists to add a marker to certain cells, so that these cells may be easily located and/or selected out from a larger cell population. The technique, described in a recent TSRI study, makes use of the tight binding of two proteins that are cheaply obtainable, but are not found in human or other mammalian cells, giving it distinct advantages over existing cell-marking techniques.
The study, "Engineering Cell Surfaces for Orthogonal Selectability," was published Dec. 13 in the online version of the chemistry journal Angewandte Chemie International Edition.
The selective addition of markers to expressed proteins has become a standard procedure in molecular biology, with the most frequently used markers being fluorescent proteins and epitope tags. Fluorescent markers such as green fluorescent protein (GFP), for example, enable the study of the subcellular localization of the protein by fluorescent microscopy, as well as the isolation of cells expressing the protein by fluorescent cell sorting. Experimenters have many methods by which to study the expression patterns of the marked protein and even isolate the cells expressing it by cell sorting. Importantly, in all these methods, one assumes that the marker does not alter the properties of the protein to which it is appended. But according to the researchers, what seems to be missing is a robust, genetic method to mark the surface of the cells themselves, so that certain cells can be easily and simply isolated from a population where they may be a minor component.
Thus, the TSRI team designed a system for the affinity selection of cells and plasma membranes based on the expression of the chitin-binding domain (ChBD) of the enzyme chitinase on the cell surface. The system is "cheap, easy and sensitive," says TSRI Institute Prof. Richard A. Lerner, who is the senior author of the new report.
The system's design is yet another crowning achievement in Lerner's long career as a research chemist, as he was the architect of the most important advance since the discovery of monoclonal antibodies a quarter century ago: the conception, design and creation of combinatorial antibody libraries, which is currently the most widely used of all libraries in the field of biochemistry and which enabled a broadening of the scope of action of the immune system. Lerner set the stage in an article published in Science in 1989, and all the advances produced in the change in combinatorial libraries derived directly or indirectly from this article. In 1991, he identified the essence of the production of antibodies without immunization, and his method remains the most efficient way to produce fully human antibodies. Moreover, Lerner has been a pioneer in the development of catalytic antibodies, a strategy to accelerate and catalyze chemical reactions for which traditional methods are not efficient.
Lerner's work has resulted in two top-selling drugs—Abbott's Humira, a treatment for inflammatory diseases such as rheumatoid arthritis, Crohn's disease and plaque psoriasis; and GlaxoSmithKline's and Human Genome Sciences' Benlysta, a treatment for lupus.
"This research is important in that it further served the antibody libraries that I invented," says Lerner. "We wanted to develop a method where we could just mix a few cells with the right targets, and a lot of cells that didn't have the targets, so that when they interact with phage, you could pick out the right cells and leave the wrong ones behind."
In the basic technique, a new gene can be added to cells within a larger DNA vector that also includes the genetic sequences for ChBD and GFP. Because the ChBD marker, in the vector, is produced in a way that anchors it to a cell's membrane, it also can serve as a powerful tool for selecting just the membrane fraction of a sample of cellular material. The ChBD molecule will be produced in such a way that it ends up being held on the outer surface of its host cell's plasma membrane—and the GFP molecule will sit just inside the membrane. The GFP serves as a visual beacon, while the ChBD serves as a handy gripping point for cell selection.
After exposing a culture of test cells to this experimental ChBD-containing vector, the scientists were able to see, via the GFP tags, which cells were expressing them, and were able to select them out easily, with high sensitivity, using magnetic beads coated with chitin. Importantly, these selected cells could produce progeny cells that seemed normal and healthy.
According to the TSRI team, this method could be important for enriching for transformants, especially when the transformation frequency is low and would facilitate combinatorial antibody selections of phage that bind to cell surfaces where, as for transformants, the target cell can be a minor component of an otherwise large population. In addition, the method would allow for the rapid affinity-based isolation of plasma membranes for biochemical studies.
"The method should be useful in a variety of applications that require separating out certain types of cells," says Lerner. "If you look at it from 30,000 feet, it is a cell-service marker, but it's really more than a marker because it slows you to selectively capture the cells that are expressed."
Lerner and his colleagues are now investigating the potential use of ChBD-based cell marking in living animals in an attempt to track the fates of selected cell types throughout an animal's lifespan.
The technology is available to researchers if they contact TSRI's Office of Technology Development, at (858) 784-8140 or through the department's webpage at http://www.scripps.edu/research/technology/contactus.html.
Other contributors to the study were first author Yingjie Peng, a postdoctoral fellow; Teresa M. Jones and Diana I. Ruiz of the TSRI Department of Chemistry; and Dae Hee Kim of the Scripps Korea Antibody Institute. The work was supported by a grant from the institute.