Genome editing by clustered regularly interspaced short palindromic sequences (CRISPR)/CRISPR‐associated protein 9 (Cas9) has revolutionized functional gene analysis and genetic improvement. While ...reporter‐assisted CRISPR/Cas systems can greatly facilitate the selection of genome‐edited plants produced via stable transformation, this approach has not been well established in seed crops. Here, we established the seed fluorescence reporter (SFR)‐assisted CRISPR/Cas9 systems in maize (Zea mays L.), using the red fluorescent DsRED protein expressed in the endosperm (En‐SFR/Cas9), embryos (Em‐SFR/Cas9), or both tissues (Em/En‐SFR/Cas9). All three SFRs showed distinct fluorescent patterns in the seed endosperm and embryo that allowed the selection of seeds carrying the transgene of having segregated the transgene out. We describe several case studies of the implementation of En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐ SFR/Cas9 to identify plants not harboring the genome‐editing cassette but carrying the desired mutations at target genes in single genes or in small‐scale mutant libraries, and report on the successful generation of single‐target mutants and/or mutant libraries with En‐SFR/Cas9, Em‐SFR/Cas9, and Em/En‐SFR/Cas9. SFR‐assisted genome editing may have particular value for application scenarios with a low transformation frequency and may be extended to other important monocot seed crops.
The seed fluorescence reporter‐assisted CRISPR/Cas9 system allows easy sorting of plants carrying or lacking the transgene. This can greatly improve working efficiency, especially for low transformation efficiency methods and generation of mutant libraries, and could be extended to other important seed crops.
The β-glucuronidase (GUS) reporter gene system is an important technique with versatile uses in the study of flower development in a broad range of species. Transcriptional and translational GUS ...fusions are used to characterize gene and protein expression patterns, respectively, during reproductive development. Additionally, GUS reporters can be used to map cis-regulatory elements within promoter sequences and to investigate whether genes are regulated post-transcriptionally. Gene trap/enhancer trap GUS constructs can be used to identify novel genes involved in flower development and marker lines useful in mutant characterization. Flower development studies primarily have used the histochemical assay in which inflorescence tissue from transgenic plants containing GUS reporter genes are stained for GUS activity and examined as whole-mounts or subsequently embedded into wax and examined as tissue sections. In addition, quantitative GUS activity assays can be performed on either floral extracts or intact flowers using a fluorogenic GUS substrate. Another use of GUS reporters is as a screenable marker for plant transformation. A simplified histochemical GUS assay can be used to quickly identify transgenic tissues.
Modern genetic approaches are powerful in providing access to diverse cell types in the brain and facilitating the study of their function. Here, we report a large set of driver and reporter ...transgenic mouse lines, including 23 new driver lines targeting a variety of cortical and subcortical cell populations and 26 new reporter lines expressing an array of molecular tools. In particular, we describe the TIGRE2.0 transgenic platform and introduce Cre-dependent reporter lines that enable optical physiology, optogenetics, and sparse labeling of genetically defined cell populations. TIGRE2.0 reporters broke the barrier in transgene expression level of single-copy targeted-insertion transgenesis in a wide range of neuronal types, along with additional advantage of a simplified breeding strategy compared to our first-generation TIGRE lines. These novel transgenic lines greatly expand the repertoire of high-precision genetic tools available to effectively identify, monitor, and manipulate distinct cell types in the mouse brain.
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•23 new driver lines and 26 new reporter lines for a wide range of applications•TIGRE2.0 reporters have viral-like transgene expression level in diverse cell types•New calcium- or voltage-sensing reporters with the former functionally characterized•Comparative analysis of new optogenetic effectors with complementary properties
An expanded toolkit of transgenic mouse lines for exploring the organization, function, and development of mammalian neural circuits.
Reverse genetics systems provide a powerful tool to generate recombinant arenavirus expressing reporters to facilitate the investigation of the arenavirus life cycle and also for the discovery of ...antiviral countermeasures. The plasmid-encoded viral ribonucleoprotein components initiate the transcription and replication of a plasmid-driven full-length viral genome, resulting in infectious virus. Thereby, this approach is ideal for the generation of recombinant arenaviruses expressing reporter genes that can be used as valid surrogates for virus replication. By splitting the small viral segment (S) into two viral segments (S1 and S2), each of them encoding a reporter gene, recombinant tri-segmented arenavirus can be rescued. Bi-reporter-expressing recombinant tri-segmented arenaviruses represent an excellent tool to study the biology of arenaviruses, including the identification and characterization of both prophylactic and therapeutic countermeasures for the treatment of arenaviral infections. In this chapter, we describe a detailed protocol on the generation and in vitro characterization of recombinant arenaviruses containing a tri-segment genome expressing two reporter genes based on the prototype member in the family, lymphocytic choriomeningitis virus (LCMV). Similar experimental approaches can be used for the generation of bi-reporter-expressing tri-segment recombinant viruses for other members in the arenavirus family.
Liquid crystal monomers (LCMs) are a large family of artificial ingredients that have been widely used in global liquid crystal display (LCD) industries. As a major constituent in LCDs as well as the ...end products of e-waste dismantling, LCMs are of growing research interest with regard to their environmental occurrences and biochemical consequences. Many studies have analyzed LCMs in multiple environmental matrices, yet limited research has investigated the toxic effects upon exposure to them. In this study, we combined in silico simulation and in vitro assay validation along with omics integration analysis to achieve a comprehensive toxicity elucidation as well as a systematic mechanism interpretation of LCMs for the first time. Briefly, the high-throughput virtual screen and reporter gene assay revealed that peroxisome proliferator-activated receptor gamma (PPARγ) was significantly antagonized by certain LCMs. Besides, LCMs induced global metabolome and transcriptome dysregulation in HK2 cells. Notably, fatty acid β-oxidation was conspicuously dysregulated, which might be mediated through multiple pathways (IL-17, TNF, and NF-kB), whereas the activation of AMPK and ligand-dependent PPARγ antagonism may play particularly important parts. This study illustrated LCMs as a potential PPARγ antagonist and explored their toxicological mode of action on the trans-omics level, which provided an insightful overview in future chemical risk assessment.
Efforts to reveal ancestral functions of auxin, a key regulator of plant growth and development, and its importance for evolution have been hampered by a fragmented picture of auxin response domains ...in early-diverging land plants. We report the mapping of auxin sensing and responses during vegetative moss development using novel reporters.
We established a moss-specific ratiometric reporter (PpR2D2) for Auxin Response Element-and AUXIN RESPONSE FACTOR-independent auxin sensing in Physcomitrella patens, and its readout during vegetative development was compared with new promoter-based GmGH3::GFPGUS and DR5revV2::GFPGUS auxin response reporters.
The ratiometric reporter responds rapidly to auxin in a time-, dose- and TRANSPORT INHIBITOR RESISTANT1/AUXIN F-BOX-dependent manner and marks known, anticipated and novel auxin sensing domains. It reveals proximal auxin sensing maxima in filamentous tissues and sensing minima in all five vegetative gametophytic stem cell types as well as dividing cells.
PpR2D2 readout is compliant with an ancestral function of auxin as a positive regulator of differentiation vs proliferation in stem cell regions. The PpR2D2 reporter is a sensitive tool for high-resolution mapping of auxin sensing, which can increase our knowledge of auxin function in early-diverging land plants substantially, thereby advancing our understanding of its importance for plant evolution.
SUMMARY
The application of DNA–protein interaction reporter assays for relational or ratiometric measurements within an experimental system is popular in biological research. However, the existing ...reporter‐based interaction assays always require special equipment, expensive chemicals, and a complicated operation. Here, we developed a DNA–protein interaction technology integrating two visible reporters, RUBY and UV‐visible GFP (eYGFPuv), which allows the expression of the cassette reporter contained cis‐acting DNA element (DE) fused upstream of TATA box and RUBY, and a constitutive promoter regulating eYGFPuv in the same construct. The interaction of transcription factor (TF) and the DE can be detected by co‐expressed the cassette reporter and TF in tobacco leaves where the cassette reporter alone serves as a control. We also revealed that eight function‐unknown bamboo AP2/ERFs interacted with the DE of ANT‐AP2R1R2 (ABE), DRE (DBE), GCC‐box (EBE), and RAV1 binding element (RBE), respectively, which are consistent with the results by dual‐luciferase reporter assays. Thus, the dual‐visible reporters offer a convenient, visible, and cost‐saving alternative to other existing techniques for DNA–protein interaction in plants.
Significance Statement
A DNA–protein interaction reporter assay has been developed by integrating two visible reporters, RUBY and eYGFPuv, which offers a convenient, visible, and cost‐saving alternative to other existing techniques for DNA–protein interaction in plants.
Perfluoralkylated substances (PFAS) such as perfluorooctanoic acid (PFOA) or perfluorooctanesulfonic acid (PFOS) are used to produce, e.g., surface coatings with water- and dirt-repellent properties. ...These substances have been shown to be hepatotoxic in rodents, and the mechanism of action is mostly attributed to the PFAS-mediated activation of the peroxisome proliferator-activated receptor alpha (PPARα). In the present study, we investigated by using luciferase-based reporter gene assays whether PFOA, PFOS and six alternative PFAS can activate, in addition to PPARα, eight other human nuclear receptors. All tested PFAS except for perfluorobutanesulfonic acid (PFBS) were able to activate human PPARα. Perfluoro-2-methyl-3-oxahexanoic acid (PMOH) and 3H-perfluoro-3-(3-methoxypropoxy) propanoic acid (PMPP) were weak agonists of human PPARγ. The other human nuclear receptors (PPARδ, CAR, PXR, FXR, LXRα, RXRα and RARα) were not affected by any PFAS tested in this study. Although PMOH was more effective than PFOA in stimulating PPARα in the transactivation assay, it was less effective in stimulating PPARα-dependent target gene expression in human HepG2 hepatocarcinoma cells. Notably, any effect observed in this in vitro study only occurred at concentrations higher than 10 μM of the respective PFAS which is in all cases several magnitudes above the average blood concentration in the Western population. Thus, the results suggest that nuclear receptor activation may only play a minor role in potential PFAS-mediated adverse effects in humans.
•Several PFAS activate human PPARα in reporter gene assays.•PFAS do not activate the human variants of PPARδ, CAR, PXR, FXR, LXRα, RXRα and RARα.•PFOA is more potent in stimulating PPARα-dependent target genes than PMOH.
Engineered recombinant viruses expressing reporter genes have been developed for real-time monitoring of replication and for mass screening of antiviral inhibitors. Recently, we reported using a ...reverse genetics system to develop the first recombinant reporter rotaviruses (RVs) that expressed NanoLuc (NLuc) luciferase. Here, we describe a strategy for developing stable reporter RVs expressing luciferase and green or red fluorescent proteins. The reporter genes were inserted into the open reading frame of NSP1 and expressed as a fusion with an NSP1 peptide consisting of amino acids 1 to 27. The stability of foreign genes within the reporter RV strains harboring a shorter chimeric NSP1-reporter gene was greater than that of those in the original reporter RV strain, independent of the transgene inserted. The improved reporter RV was used to screen for neutralizing monoclonal antibodies (MAbs). Sequence analysis of escape mutants from one MAb clone (clone 29) identified an amino acid substitution (arginine to glycine) at position 441 in the VP4 protein, which resides within neutralizing epitope 5-1 in the VP5* fragment. Furthermore, to express a native reporter protein lacking NSP1 amino acids 1 to 27, the 5'- and 3'-terminal region sequences were modified to restore the predicted secondary RNA structure of the NSP1-reporter chimeric gene. These data demonstrate the utility of reporter RVs for live monitoring of RV infections and also suggest further applications (e.g., RV vaccine vectors, which can induce mucosal immunity against intestinal pathogens).
Development of reporter RVs has been hampered by the lack of comprehensive reverse genetics systems. Recently, we developed a plasmid-based reverse genetics system that enables generation of reporter RVs expressing NLuc luciferase. The prototype reporter RV had some disadvantages (i.e., the transgene was unstable and was expressed as a fusion protein with a partial NSP1 peptide); however, the improved reporter RV overcomes these problems through modification of the untranslated region of the reporter-NSP1 chimeric gene. This strategy for generating stable reporter RVs could be expanded to diverse transgenes and be used to develop RV transduction vectors. Also, the data improve our understanding of the importance of 5'- and 3'-terminal sequences in terms of genome replication, assembly, and packaging.
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