Winners of Addgene’s rDNA Technology Award Show Innovation in Plasmid Design
Addgene, a Cambridge-based nonprofit that helps scientists share recombinant DNA tools known as plasmids, announced its most recent Innovation Award winners (http://www.addgene.org/awards). The Recombinant DNA (rDNA) Technology Award is the second in a series of three awards that Addgene is sponsoring this year. The rDNA Technology Award is a unique funding opportunity recognizing laboratories developing new DNA-based technologies to address important biomedical questions. Winners were chosen based on the technical design and approach of their projects.
Dr. Robinson’s group is designing new methods for heterologous protein expression in yeast. Using G-Protein Coupled Receptors (GPCRs) as the proof of concept in their studies, the Robinson group is developing a high-throughput approach to expressing functional proteins and methodologies to determine protein localization. GPCRs are a family of membrane proteins that have proven to be excellent targets for drug design for a myriad of diseases. GPCRs are ubiquitous in the sense that they are expressed in a variety of cell types and can be used to transmit different stimuli from the extracellular environment. One current roadblock in the field is the ability to obtain high enough protein yields for studying these membrane-bound proteins.
Zack Britton and Carissa Young, graduate students in the Robinson laboratory leading these projects explain, “The system is not limited to membrane proteins, as the design of the plasmids is versatile.” With these novel expression systems in place, the Robinson lab hopes that other researchers will take advantage of the design flexibility and use them to find optimal conditions for expression of additional proteins of interest.
Award winners for this opportunity were chosen both on the ability of the plasmid design to address important technical barriers in the researcher’s respective field and the adaptability of the technology to a larger audience.
Dr. Richard Steinman at the University of Pittsburgh is developing a set of plasmids that can be used to assay for the global transcriptome of cells between two states. In this project, Dr. Steinman is trying to assess the genes responsible for the exit from quiescence or “awakening” of dormant breast cancer cells.
“Roughly a quarter of women with breast cancer suffer from metastatic recurrence of disease after treatment,” explains Dr. Steinman, “What compels this project (specifically) in the area of cancer, is that the problem of recurrent disease is as daunting as always.”
Using fluorescent markers to “tag” cells that are either dormant or actively dividing, Dr. Steinman is able to couple the expression markers with time-limited metabolic labeling to determine the profile of the cells as they exit quiescence. With simple manipulations of the plasmids, researchers can adapt the system to look at the global transcriptional profile of any two biologically relevant states. For example, the plasmids can be used to understand the genes responsible for specific stages in stem cell differentiation.
Laboratories that applied for this award not only showed their dedication to progressive science, but also designing tools to better equip others to achieve their goals.
Dr. Steinman is encouraged by opportunities that focus on advancing experimental techniques, “NIH is increasingly recognizing innovative approaches, however just getting things to the point of principle is tough; so there’s a gap in this (funding) area.”
Julie Hunkapiller, a graduate student at UCSF in award-winner Dr. Jeremy Reiter’s lab agrees that (funding) opportunities for technology-driven projects in the biological sciences is minimal. “We’re really excited about this award, as it’s been hard to find anything similar out there (that focuses on the technology).”
The Reiter lab recently developed a method for efficient and targeted modification of genes in mouse embryonic stem cells (ESCs). Floxin (for Flanked lox site insertion) is based on a plasmid that can be used to insert a researcher’s desired DNA sequence at specific loci in the mouse genome. Using a shuttle vector known as pFloxin, the technology is compatible with gene trap cell lines, where the site of insertion has already been characterized. Floxin also relies on Cre-mediated recombination, which has proven to be much more efficient in generating targeted cells.
The Reiter lab, realizing the potential of the technology, is currently trying to adapt the Floxin system for use in human ESCs. “In human ESCs, we hope to be able to make specific disease mutations and then look at how these mutations affect early human development in different lineages,” explains Ms. Hunkapiller.
While plasmids are highly diverse, this award also helps to highlight the ubiquitous nature of this important biological tool.
Dr. Joseph Wade, an award winner at the Wadsworth Center in Albany, is building a two-tiered plasmid system to help researchers tag proteins in bacteria and target them for rapid degradation.
“There are multiple challenges with getting DNA into bacteria effectively,” explains Dr. Wade, “there’s the issue of selection (finding bacteria which contain your gene of interest) and there’s the issue of scarring the chromosome.”
The Wade lab is taking advantage of the Thymidylate Synthase gene, thyA, to overcome some of these hurdles. The gene allows for both selection of the incorporated gene into the bacterial genome and then counter-selection, where the marker itself is recombined out, leaving the bacterial genome “scar-free,” but both the gene and tag intact. With the ability to incorporate the epitope tags efficiently into the genome, this will also allow researchers to study proteins in bacteria using commonly available antibodies.
Combined with the ability to target proteins for degradation, Dr. Wade hopes to use the system for genomic studies looking at the role of Rho, a transcriptional termination protein, and how it functions in regulating transcription in bacteria. The system is very flexible, in that both the epitope tags and the degron tag can be used by researchers studying both essential and non-essential genes in various species.
From bacteria to yeast to mammalian systems, this award emphasizes the utility of plasmids in the life sciences and the ongoing need for new technologies to help address important biomedical questions.
Founded in 2004 as a hub for scientific sharing and to promote biological and medical discoveries, Addgene has ties to many MA-based universities including MIT, Tufts, and Harvard. Addgene stores, archives, and distributes plasmids, the gold currency for recombinant DNA. Plasmids are used by researchers worldwide to study how genes function. With the ability to distribute high-quality plasmids in a matter of days, Addgene provides a valuable service to the academic research community.
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