Genetic Engineering & Biotechnology News

JUN15 2018

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16 | JUNE 15, 2018 | Genetic Engineering & Biotechnology News | their insights, many of which are summarized in this article. Signatures of Aging Some of the presenters at Single Cell Genomics 2017 demonstrated how single-cell RNA sequencing data from primary cells could be used to illuminate genetic and tran- scriptional processes that occur in aging human tissue. For example, Stephen Quake, Ph.D., a professor of bioengineer- ing at Stanford University and co-president of the Chan Zuckerberg BioHub, discussed his work with Seung K. Kim, Ph.D., a professor of developmental biology at Stanford. The Quake and Kim laboratories used single-cell transcrip- tomics to evaluate the effects of aging on the pancreas. In this work, 2544 human pancreas cells spanning six decades of age were analyzed. Entire transcriptomes were compared. Findings showed that transcriptional entropy increased globally with age. Cells were diverging, transcriptionally. With age, the muta- tional load increased in ways that were characteristic of in- creasing oxidative damage. This mutational burden increase was unexpected and challenging to measure at the single-cell level. Another all-Stanford collaboration, between Dr. Quake's group and the laboratory of Liqun Luo, Ph.D., a professor of biology, aimed to understand if single-cell transcriptomic measurements really reflected physiological ground truth as cell biologists have defined it for decades in terms of mea- suring cellular identity. In this collaboration, the research focused on Drosophila olfactory projection neurons (PNs). These are among the best-characterized neuronal types. Different PN classes target dendrites to distinct olfactory glomeruli. PNs occur in different clusters; cells of the same class exhibit identical anatomi- cal and physiological properties. These physiologically distinct cell types, Dr. Quake indicated, were identified by analyzing transcriptomes. Also discovered were new transcription factors that are involved in the way the cells wire up together and decide which cells they are going to rec- ognize. "The first challenges in the single-cell genomics field were to develop the technical methods and prove they worked," said Dr. Quake. "Now that that has been accomplished, new ques- tions have emerged." For example: What is the best science one can do with these approaches? "We find that these technologies represent a valuable tool in the arsenal one brings to big biological questions," Dr. Quake continued. "More and more we will see single-cell approaches used to complement conventional approaches to understanding how cells evolve and what their roles are in the human organism." Current methods for single-cell genomics include cell sort- ing, microfluidic methods incorporating fabricated valves, and microfluidic emulsion-based approaches. A new method, Microwell-seq, a high-throughput and low-cost scRNA-seq platform, was recently described by a group from Zhejiang University. Different methods present different strengths and are suited to the resolution of different of problems. One Pot, Two "Omes" Hundreds of transcripts from a single cell can be measured in high throughput. This kind of transcriptomic analysis could be complemented, scientists hope, by high-throughput multiplexed single-cell proteomic techniques. The difficulty, however, is that the number of proteins that can be measured per cell is limited using mass spectrometry. Progress toward transcriptomic/proteomic complemen- tarity has been reported by Savas Tay, Ph.D., an associate professor at the Institute of Molecular Engineering and the Institute for Genomics and System Biology at the University of Chicago. In Dr. Tay's laboratory, a microfluidic single-cell technology is being developed that could potentially measure hundreds to thousands of individual proteins and complexes all in the same pot, along with the transcriptome. Two antibodies tagged with short oligos of a known sequence target a protein. After the antibody binds to the proteins, cells are lysed, the oligos are sequenced, and the binding abundance in the cell is determined. This proximal ligation assay approach increases sensitivity and minimizes antibody noise and off-target effects. Protein complexes can also be measured; 50 antibody-oli- go pairs could measure up to 2500 complexes. The sequenc- ing readout makes the technology compatible with RNA sequencing. In a single droplet, both the transcriptome and proteome are measured. "In single-cell research, you want to measure as many species as possible," noted Dr. Tay. "We would like to con- tinue using droplet-based sequencing and develop protocols that will label the cells spatially and temporally to continue to use our high-throughput methods." Current technologies do not allow the measurement of temporal evolution of cells in a high-throughput multiplex fashion; endpoint measurements do not provide temporal information. Much effort is required to understand temporal aspects through static but still computationally heavy mea- surements. Longitudinal cell-imaging experiments to study temporal and spatial patterns are a cornerstone of biology, but the low- resolution protocols are limited by pipetting requirements. A new high-throughput microfluidic culture and live-cell analy- sis system is under development to keep cells alive and expose them to reagents in a spatially and temporally controlled fash- ion to mimic the native environment. The device performs mil- OMICS Microfluidic single-cell technology is being developed that could potentially measure thousands of individual proteins and protein complexes along with the transcriptome. Single-Cell Analysis for a More Perfect Cell Biology Continued from page 1 " More and more we will see single-cell approaches used to complement conventional approaches to understanding how cells evolve and what their roles are in the human organism." —Stephen Quake, Ph.D. Stanford University and Chan Zuckerberg BioHub

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