Using the 2A Protein Coexpression System: Multicistronic 2A Vectors Expressing Gene(s) of Interest and Reporter Proteins

2018 ◽  
pp. 31-48 ◽  
Author(s):  
Garry A. Luke ◽  
Martin D. Ryan
Keyword(s):  
Reporter Proteins

scholarly journals FAST Is a Survival Protein That Senses Mitochondrial Stress and Modulates TIA-1-Regulated Changes in Protein Expression

2004 ◽  
Vol 24 (24) ◽  
pp. 10718-10732 ◽  
Author(s):  
Wei Li ◽  
Maria Simarro ◽  
Nancy Kedersha ◽  
Paul Anderson
Keyword(s):  
Protein Synthesis ◽  
Rna Interference ◽  
Stress Response ◽  
Mitochondrial Membrane ◽  
Binding Domain ◽  
Outer Mitochondrial Membrane ◽  
Mitochondrial Stress ◽  
Inhibitors Of Apoptosis ◽  
Reporter Proteins ◽  
Induced Apoptosis

ABSTRACT The Fas-activated serine/threonine phosphoprotein (FAST) is tethered to the outer mitochondrial membrane, where it interacts with BCL-XL (17). Here we show that RNA interference-mediated knockdown of endogenous FAST results in apoptosis, whereas overexpressed recombinant FAST inhibits Fas- and UV-induced apoptosis, indicating that FAST is a survival protein. The antiapoptotic effects of FAST are regulated by interactions with the translational silencer TIA-1: a FAST mutant lacking its TIA-1-binding domain does not inhibit apoptosis, and overexpressed recombinant TIA-1 inhibits the antiapoptotic effects of FAST. Because the antiapoptotic effects of FAST require ongoing protein synthesis, we hypothesized that FAST might function by preventing TIA-1-mediated silencing of mRNAs encoding inhibitors of apoptosis. Consistent with this hypothesis, FAST promotes the expression of cotransfected reporter proteins, a process that requires its TIA-1-binding domain and is inhibited by overexpressed recombinant TIA-1. More compellingly, recombinant FAST increases the expression of endogenous cIAP-1 and XIAP, but not GAPDH, in transfected HeLa cells. Because FAST is released from mitochondria in cells undergoing Fas- or UV-induced apoptosis, we propose that FAST serves as a sensor of mitochondrial stress that modulates a TIA-1-regulated posttranscriptional stress response program.

scholarly journals Cell Cultures for Virology: Usability, Advantages, and Prospects

2020 ◽  
Vol 21 (21) ◽  
pp. 7978
Author(s):  
Alexander A. Dolskiy ◽  
Irina V. Grishchenko ◽  
Dmitry V. Yudkin
Keyword(s):  
Cell Lines ◽  
Cell Cultures ◽  
Virus Isolation ◽  
Virus Detection ◽  
Clinical Samples ◽  
Laboratory Setting ◽  
Reporter Cell ◽  
Isolation And Identification ◽  
Reporter Systems ◽  
Reporter Proteins

Virus detection in natural and clinical samples is a complicated problem in research and diagnostics. There are different approaches for virus isolation and identification, including PCR, CRISPR/Cas technology, NGS, immunoassays, and cell-based assays. Following the development of genetic engineering methods, approaches that utilize cell cultures have become useful and informative. Molecular biology methods allow increases in the sensitivity and specificity of cell cultures for certain viruses and can be used to generate reporter cell lines. These cell lines express specific reporter proteins (e.g., GFP, luciferase, and CAT) in response to virus infection that can be detected in a laboratory setting. The development of genome editing and synthetic biology methods has given rise to new perspectives regarding the design of virus reporter systems in cell cultures. This review is aimed at describing both virology methods in general and examples of the development of cell-based methods that exist today.

scholarly journals Motor-driven motility of fungal nuclear pores organizes chromosomes and fosters nucleocytoplasmic transport

2012 ◽  
Vol 198 (3) ◽  
pp. 343-355 ◽  
Author(s):  
Gero Steinberg ◽  
Martin Schuster ◽  
Ulrike Theisen ◽  
Sreedhar Kilaru ◽  
Andrew Forge ◽  
...  
Keyword(s):  
Nuclear Lamina ◽  
Ustilago Maydis ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Nuclear Pore Complexes ◽  
Nuclear Pores ◽  
Gfp Reporter ◽  
Import And Export ◽  
Reporter Proteins ◽  
Pore Complexes

Exchange between the nucleus and the cytoplasm is controlled by nuclear pore complexes (NPCs). In animals, NPCs are anchored by the nuclear lamina, which ensures their even distribution and proper organization of chromosomes. Fungi do not possess a lamina and how they arrange their chromosomes and NPCs is unknown. Here, we show that motor-driven motility of NPCs organizes the fungal nucleus. In Ustilago maydis, Aspergillus nidulans, and Saccharomyces cerevisiae fluorescently labeled NPCs showed ATP-dependent movements at ∼1.0 µm/s. In S. cerevisiae and U. maydis, NPC motility prevented NPCs from clustering. In budding yeast, NPC motility required F-actin, whereas in U. maydis, microtubules, kinesin-1, and dynein drove pore movements. In the latter, pore clustering resulted in chromatin organization defects and led to a significant reduction in both import and export of GFP reporter proteins. This suggests that fungi constantly rearrange their NPCs and corresponding chromosomes to ensure efficient nuclear transport and thereby overcome the need for a structural lamina.

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