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Summary of the September 2015 Issue of BioTechniques

This month in BioTechniques, there are new methods for:

  • cell free protein synthesis
  • screening of protein libraries using FACS
  • gene synthesis without PCR
  • preparation of adherent cells for flow cytometry
  • fixing and imaging GFP in tissues
  • determining the impact of vascular endothelial cells on cytomegalovirus dissemination

In addition to these new peer-reviewed methods articles, September will also feature a Tech News article examining the growth of ligation-free cloning and gene assembly methods, and how these approaches are changing the ways in which researchers approach gene function studies in the lab today.

Cell-free protein synthesis allows researchers to rapidly generate functional proteins independent of cell culture. Although advances in eukaryotic lysates have increased the amount of protein that can be produced, nuances between translation systems leads to variability in protein production. To help overcome this problem, in the September issue researchers from Arizona State University compared the relative yield and template requirements for three commonly used commercial cell-free translation systems: wheat germ extract (WGE), rabbit reticulocyte lysate (RRL), and HeLa cell lysate (HCL). Their results provide a general guide for researchers interested in using cell-free translation to generate recombinant protein for biomedical applications.

Keywords: protein analysis; cell free protein synthesis; proteomics; protein analysis

Iterative screening of expressed protein libraries using fluorescence-activated cell sorting (FACS) typically involves culturing the pooled clones after each sort. If cell viability is compromised by sorting conditions or presence of the target protein, alternative methods such as rescue PCR are time-consuming, laborious and can introduce amplification bias. In September, researchers from the University of Houston present an optimized protocol using commercially available reagents to directly recover plasmid DNA from sorted cells for transformation. The authors tested the protocol with two different screening systems in which less than 10% of sorted cells survive culturing and demonstrated that >60% of the sorted cell population was recovered, leading to efficient functional enrichment after just one round of FACS.

Keywords: FACS; protein libraries; protein analysis; plasmid DNA recovery

Current gene synthesis methods often incorporate a PCR amplification step. Amplification, however, can cause stochastic sampling effects that propagate errors in gene synthesis or lower variability when applied towards the construction of randomized libraries. Researchers from the University of Colorado at Boulder have developed a simple DNA polymerase-based gene synthesis reaction, Polymerase Step Reaction (PSR), which assembles DNA oligonucleotides in a unidirectional fashion without requiring amplification. This approach will enable less off-target variability to be introduced into the gene synthesis process.

Keywords: gene synthesis; amplification; linear amplification; error-free DNA synthesis

The optimization of cell preparations continues to be an important aspect of flow cytometry methods development. In the September issue, researchers from Saudi Arabia describe a new protocol that bypasses the washing, centrifugation, and transfer between plates that are involved in standard multistep protocols for preparing adherent cells for flow cytometry. The method was validated using six adherent cell lines and the results have been confirmed using two different flow cytometry instruments. In addition, the authors used the protocol for estimating apoptosis, mitochondrial membrane potential, reactive oxygen species and autophagy in response to pure compounds as well as plant and bacterial extracts.

Keywords: flow cytometry; adherent cells; flow cytometry applications

Green fluorescent protein and its derivatives are routinely employed as surrogate markers for gene expression and lineage tracing in genetically engineered mice. Tissues from these mice are commonly formalin fixed and paraffin embedded for histological studies. However, this results in inactivation of the natural fluorescence of these proteins, requiring their detection by immunological techniques. This month, scientists from Massachusetts General Hospital in Boston, MA present an ethanol fixation protocol that allows for the direct visualization of the natural fluorescence of reporter proteins while maintaining excellent tissue histology. The authors demonstrate the utility of this method for visualization of green and red fluorescent proteins in a wide range of murine tissues using both cytoplasmic and membrane localized fluorescent reporter proteins.

Keywords: GFP; FFPE; immunological techniques; histology

Infection of vascular endothelial cells is assumed to contribute to dissemination of human cytomegalovirus (HCMV). Investigation of virus-host interactions in endothelial cells such as human umbilical vein endothelial cells (HUVECs) is limited due to the low maximal passage numbers of these primary cells. This month, researchers from Germany have tested a conditionally immortalized endothelial cell line (HEC-LTT) and a permanent cell line (EA.hy926) for their susceptibility to HCMV infection. Both cell lines resembled HUVECs in that they allowed for entry and immediate early protein expression of highly endotheliotropic HCMV strains but not of poorly endotheliotropic strains, rendering them suitable for analysis of the viral entry mechanism in endothelial cells. The implementation of permanent HEC-LTTs and EA.hy926 cell lines in HCMV research will facilitate long-term approaches that are not feasible in primary HUVECs.

Keywords: cell analysis; cell culture; CMV; primary cells

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