Hypoxia has been reported to promote tumor progression and metastasis in murine models, and patients with hypoxic tumors have a worse prognosis. Besides its effect on cancer, normal processes like ...embryogenesis, or other pathologies such as ischemia, depend on hypoxia-regulated mechanisms. Given the degradable nature of HIF-1/2α in the presence of oxygen, defining the role of hypoxia in modeling biological processes becomes challenging when a cell enters oxygen-rich regions within a tissue. Here, we describe a unique approach to permanently mark cells that experience hypoxia with a fluorescent protein switch that is maintained even after a cell is reoxygenated. This method consists of a dual-viral delivery system that can be transduced into any mammalian cell line.
Massively parallel reporter gene assays are key tools in regulatory genomics but cannot be used to identify cell-type-specific regulatory elements without performing assays serially across different ...cell types. To address this problem, we developed a single-cell massively parallel reporter assay (scMPRA) to measure the activity of libraries of cis-regulatory sequences (CRSs) across multiple cell types simultaneously. We assayed a library of core promoters in a mixture of HEK293 and K562 cells and showed that scMPRA is a reproducible, highly parallel, single-cell reporter gene assay that detects cell-type-specific cis-regulatory activity. We then measured a library of promoter variants across multiple cell types in live mouse retinas and showed that subtle genetic variants can produce cell-type-specific effects on cis-regulatory activity. We anticipate that scMPRA will be widely applicable for studying the role of CRSs across diverse cell types.
Adeno-associated virus (AAV)-mediated CRISPR-Cas9 editing holds promise to treat many diseases. The immune response to bacterial-derived Cas9 has been speculated as a hurdle for AAV-CRISPR therapy. ...However, immunological consequences of AAV-mediated Cas9 expression have thus far not been thoroughly investigated in large mammals. We evaluate Cas9-specific immune responses in canine models of Duchenne muscular dystrophy (DMD) following intramuscular and intravenous AAV-CRISPR therapy. Treatment results initially in robust dystrophin restoration in affected dogs but also induces muscle inflammation, and Cas9-specific humoral and cytotoxic T-lymphocyte (CTL) responses that are not prevented by the muscle-specific promoter and transient prednisolone immune suppression. In normal dogs, AAV-mediated Cas9 expression induces similar, though milder, immune responses. In contrast, other therapeutic (micro-dystrophin and SERCA2a) and reporter (alkaline phosphatase, AP) vectors result in persistent expression without inducing muscle inflammation. Our results suggest Cas9 immunity may represent a critical barrier for AAV-CRISPR therapy in large mammals.
Neutralizing antibody (NAb) assays for human immunodeficiency virus (HIV) are used to study the immune response in infected individuals, to examine monoclonal antibodies and viral diversity, and to ...judge the potential value of candidate vaccine immunogens in preclinical and clinical trials. An important aspect of these efforts is an ability to achieve and document equivalent assay performance across multiple laboratories. Recent advances in assay technology have led to major improvements in how HIV NAbs are measured. Stable cell lines containing HIV Tat-regulated reporter genes are now available that permit rapid, sensitive and reproducible measurements of virus neutralization after a single round of infection in a high throughput format.Moreover, these assays may be used with molecularly cloned Env-pseudotyped viruses for greater reagent stability and traceability.A luciferase (Luc) reporter gene assay performed in TZM-bl (JC53bl-13) cells was recently optimized and many of its performance parameters have been validated. This assay has become the main endpoint neutralization assay used by the NIH-sponsored HIV Vaccine Trials Network and by a growing number of laboratories worldwide.
In order to analyze how a signal transduction network converts cellular inputs into cellular outputs, ideally one would measure the dynamics of many signals within the network simultaneously. We ...found that, by fusing a fluorescent reporter to a pair of self-assembling peptides, it could be stably clustered within cells at random points, distant enough to be resolved by a microscope but close enough to spatially sample the relevant biology. Because such clusters, which we call signaling reporter islands (SiRIs), can be modularly designed, they permit a set of fluorescent reporters to be efficiently adapted for simultaneous measurement of multiple nodes of a signal transduction network within single cells. We created SiRIs for indicators of second messengers and kinases and used them, in hippocampal neurons in culture and intact brain slices, to discover relationships between the speed of calcium signaling, and the amplitude of PKA signaling, upon receiving a cAMP-driving stimulus.
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•Clustering fluorescent sensors at points in cells enables many to be imaged at once•Modular reagent design allows existing sensors to be easily adapted to cluster•Such “signaling reporter islands” (SiRIs) are safe and robust in cells and in vivo•SiRIs reveal relationships between components of signal transduction networks
Simultaneous signals such as second messengers and kinase activities, within a single cell, can be captured through the fusion of fluorescent reporters to pairs of self-assembling peptides to generate stable signaling reporter islands.
We describe the production of a human induced pluripotent stem cell (iPSC) line, SFCi55-ZsGr, that has been engineered to express the fluorescent reporter gene, ZsGreen, in a constitutive manner. The ...CAG-driven ZsGreen expression cassette was inserted into the AAVS1 locus and a high level of expression was observed in undifferentiated iPSCs and in cell lineages derived from all three germ layers including haematopoietic cells, hepatocytes and neurons. We demonstrate efficient production of terminally differentiated macrophages from the SFCi55-ZsGreen iPSC line and show that they are indistinguishable from those generated from their parental SFCi55 iPSC line in terms of gene expression, cell surface marker expression and phagocytic activity. The high level of ZsGreen expression had no effect on the ability of macrophages to be activated to an M(LPS + IFNγ), M(IL10) or M(IL4) phenotype nor on their plasticity, assessed by their ability to switch from one phenotype to another. Thus, targeting of the AAVS1 locus in iPSCs allows for the production of fully functional, fluorescently tagged human macrophages that can be used for in vivo tracking in disease models. The strategy also provides a platform for the introduction of factors that are predicted to modulate and/or stabilize macrophage function.
This article is part of the theme issue ‘Designer human tissue: coming to a lab near you’.
Highlights • Nuclear receptor activities of OPFRs were studied by in vitro reporter gene assays. • TPhP and TCP acted as ERα/β and PXR agonists as well as AR and GR antagonists. • TDCPP was the most ...potent AR antagonist among the tested OPFRs. • None of the OPFRs had TRα/β, RARα, RXRα, or PPARα/γ activity. • Several OPFRs might be endocrine disruptors via ERα/β, AR, GR, or PXR.
Abstract Zika virus (ZIKV) infection is a major public health problem with severe human congenital and neurological anomalies. The screening of anti-ZIKV compounds and neutralizing antibodies needs ...reliable and rapid virus-based assays. Here, we described a convenient method leading to the rapid production of molecular clones of ZIKV. To generate a molecular clone of ZIKV strain MR766NIID , the viral genome was directly assembled into Vero cells after introduction of four overlapping synthetic fragments that cover the full-length genomic RNA sequence. Such strategy has allowed the production of a recombinant ZIKV expressing the GFP reporter gene that is stable over two culturing rounds on Vero cells. Our data demonstrate that the ZIKV reporter virus is a very reliable GFP-based tool for analyzing viral growth and measuring the neutralizing antibody as well as rapid screening of antiviral effect of different classes of inhibitors.
The APETALA2 (AP2) transcription factors (TFs) play crucial roles in regulating development in plants. However, a comprehensive analysis of the AP2 family members in a valuable Chinese herbal orchid,
..., or in other orchids, is limited. In this study, the 14 DoAP2 TFs that were identified from the
genome and named DoAP2-1 to DoAP2-14 were divided into three clades: euAP2, euANT, and basalANT. The promoters of all
genes contained
-regulatory elements related to plant development and also responsive to plant hormones and stress. qRT-PCR analysis showed the abundant expression of
,
,
,
and
genes in protocorm-like bodies (PLBs), while
,
,
,
,
and
expression was strong in plantlets. In addition, the expression of some
genes was down-regulated during flower development. These results suggest that
genes may play roles in plant regeneration and flower development in
. Four
genes (
from euAP2,
-
from euANT, and
and
from basal ANT) were selected for further analyses. The transcriptional activation of DoAP2-1, DoAP2-2, DoAP2-6 and DoAP2-11 proteins, which were localized in the nucleus of
mesophyll protoplasts, was further analyzed by a dual-luciferase reporter gene system in
leaves. Our data showed that pBD-DoAP2-1, pBD-DoAP2-2, pBD-DoAP2-6 and pBD-DoAP2-11 significantly repressed the expression of the LUC reporter compared with the negative control (pBD), suggesting that these DoAP2 proteins may act as transcriptional repressors in the nucleus of plant cells. Our findings on
genes in
shed light on the function of
genes in this orchid and other plant species.
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