Genetic Engineering & Biotechnology News

OCT15 2017

Genetic Engineering & Biotechnology News (GEN) is the world's most widely read biotech publication. It provides the R&D community with critical information on the tools, technologies, and trends that drive the biotech industry.

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Genetic Engineering & Biotechnology News | GENengnews.com | OCTOBER 15, 2017 | 19 environment including tunnels and other ob- jects. As they got excited they were turning on transcription processes in specific neurons involved in making memories." In collaboration with Fred H. Gage, Ph.D., at the Salk Institute for Biologi- cal Studies and others, the investigators discovered major changes in the neuronal transcriptome program including upregula- tion of immediate early genes (IEG) such as FOS, ARC, and EGR1. Dr. Lasken summa- rizes, "The IEG expression of single nuclei that were derived from activated neurons was consistent with known responses to behavioral experiences. Analysis of the entire transcriptome revealed extensive up- regulation of genes involved with synapse ion channels and other features suspected to play a role in memory formation. This is the first look at the entire transcriptional profile in individual neurons activated by external stimuli, and is a critical step in ul- timately discovering how a memory is cap- tured and stored." Dr. Lasken says nuclear transcriptomes also can be obtained from postmortem hu- man brain tissue stored at −80 °C, making brain archives accessible for RNA-seq from individual neurons. Further, snRNA-seq can be applied to other types of tissue as well as individual cells including eggs, circulating T cells, and cultured cells. He concludes, "I believe snRNA-seq is a robust technique for examining heterogeneous transcriptional profiles from a variety of samples. This is quite a breakthrough!" Emerging Applications and Instrumentation With the fast-paced progress developing in single-cell genomics, it is no surprise that instrumentation is expanding in parallel with applications. BD Genomics, a division of BD (Becton Dickinson and Company), recently presented information on their current and emerging platforms and applications for sin- gle-cell genomics and how they have begun to apply these to the study of solid tumors and liquid biopsies. John Mikszta, Ph.D., director, genomic sciences at the company explains, "There are three key challenges in the field. The first is the complex and challenging work- flow of preparing libraries for next-genera- tion sequencing (NGS). Often, library prep is difficult and expensive. To address this problem, we developed the BD CLiC sys- tem that miniaturizes library prep reactions to enable cost savings for the end-user. The system has scalable throughput and can process 24–384 samples per run in a fully automated fashion; the user simply hits 'go' and walks away." A second challenge is applying single cell genomics to clinical oncology research, such as the study of tumor heterogeneity, in order to distinguish all cells in the tu- mor microenvironment. "For single-cell ge- nomic analysis of solid tumors, we provide reagents and methods to dissociate tumors into single-cell suspensions. These suspen- sions are then amenable to cell-surface pro- tein analysis by BD FACS flow cytometry platforms and single-cell gene-expression analysis with a product called BD Precise. Individual cells are sorted into 96 well plates prefilled with reagents for single cell gene expression. The use of molecular in- dexing eliminates PCR bias and improves accuracy by >100-fold." The company recently developed the BD Rhapsody platform and reagents, according to Dr. Mikszta (Figure 3). "This allows eval- uation of single-cell gene expression from hundreds to thousands of cells. The platform has a simple one-day workflow that allows substantial cost reductions per sample." Further, in collaboration with clinical re- searchers at leading academic medical cen- ters, the company is developing a system for rare-cell enrichment based on magnetic depletion and acoustic focusing. This cell- enrichment system is placed upstream of a traditional BD FACS sorter to enable circu- lating tumor cell capture for downstream single-cell genomics. Dr. Mikszta asserts, "While the rare- cell-enrichment technology used in these experiments is still in early development, we already see that it will offer a powerful platform for rare-cell isolation and charac- terization." OMICS • Utilize the proper freezing technique for each cell line. Most require slow cooling, though some (e.g. stem cells) prefer ultra- rapid freezing. • Liquid vs. vapor phase nitrogen storage — while liquid phase storage allows for lower temperatures and a longer response time to storage issues, it has a higher risk of contamination and cracked/bursting vials. Vapor phase nitrogen is a great alternative to store cells and provide lower risk for contamination. • To safeguard against loss, store important stocks in more than one location and thaw one vial to assess cell viability soon after cryopreservation. Cell Growth Keeping cells healthy by mimicking their natural environment is no easy task. Vigi- lance is key. Under the hood: • Never work with more than one cell line at a time and don't share bottles of media between cell lines. • Prepare and filter media on the same day it will be used. • Pay special attention to serum quality. When possible, buy sera in bulk and test each batch to ensure it supports cell growth. • To combat genotypic and phenotypic instability, characterize cells regularly and replace from frozen stock frequently. Detecting microbial contamination: • Screen for infections (especially the most common, mycoplasma, which doesn't produce visible signs of contamination) at regular intervals. • Don't routinely use antibiotics to control contamination, as they can mask signs of infection. In antibiotic-free media, signs of bacterial, yeast, and fungal infection include cloudiness as well as color and pH changes. Cell Analysis Monitoring and quantifying cell health and function is imperative to yield biologically meaningful data. Cell counting: • Count your cells often to examine your cell culture. • The Coulter principle is the most precise method for counting cells, where cells flow, one by one, through an aperture within an electrical sensor. • Data generated by Coul - ter principle-based cell counters not only deliver precise cell counts but also display average cell size and population- distribution information. • Another benefit of using a Coulter principle-based cell counter is that you'll also obtain a histogram of the size distri- bution of your cell population, which can tell you how monodisperse your sample is. • Make sure the cells you are counting are well mixed, or you'll end up with local vari- ations in cell density that could introduce error into your cell counts. • Use a cell counter that doesn't involve loading a chamber, and you won't need to worry about air bubbles. • Transferring cell samples can introduce errors. Using a handheld, automated cell counter without leaving the culture hood is the best way to avoid variability and obtain an accurate snapshot of your culture. • Check your instrument specifications when preparing a sample and make sure your cell diameter and starting dilution are compatible with your counting method. Recommended Reading 1. Geraghty RJ et al. Guidelines for the use of cell lines in biomedical research. Br J Cancer. 111:1021-1046 (2014). 2. Yu M et al. A resource for cell line authentica- tion, annotation, and quality control. Nature. 520:307-311 (2015). Quality is paramount when it comes to cell culture supplies. MilliporeSigma has over 20,000 proven tools to help you cultivate consistency: • Thousands of authenticated cell lines and primary cells • High-flow rate sterile filtration systems • Application-tested cell freezing solutions • High-quality liquid media, serum, and supplements/growth factors • A diverse line of cultureware and spe- cialty culture inserts and plates For all your cell culture needs, visit SigmaAldrich.com/cellculture Advertiser retains sole responsibility for content Destroyed culture due to massive microbial outgrowth and antibiotic resistant organisms

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