Heterogeneity and genomic loci of ubiquitous Cre reporter transgenes in zebrafish

The most-common strategy for zebrafish Cre/lox-mediated lineage labeling experiments combines ubiquitously expressed, lox-based Switch reporter transgenes with tissue-specific Cre or 4-OH-Tamoxifen-inducible CreERT2 driver lines. Although numerous Cre driver lines have been produced, only a few broadly expressed Switch reporters exist in zebrafish and their generation by random transgene integration has been challenging due to position-effect sensitivity of the lox-flanked recombination cassettes. Here, we compare commonly used Switch reporter lines for their recombination efficiency and reporter expression pattern during zebrafish development. Using different experimental setups, we show that ubi:Switch and hsp70l:Switch outperform current generations of two additional Switch reporters due to favorable transgene integration sites. Our comparisons also document preferential Cre-dependent recombination of ubi:Switch and hsp70l:Switch in distinct zebrafish tissues at early developmental stages. To investigate what genomic features may influence Cre accessibility and lox recombination efficiency in highly functional Switch lines, we mapped these transgenes and charted chromatin dynamics at their integration sites. Our data documents the heterogeneity among lox-based Switch transgenes towards informing suitable transgene selection for lineage labeling experiments. Our work further proposes that ubi:Switch and hsp70l:Switch define genomic integration sites suitable for universal transgene or switch reporter knock-in in zebrafish.

Stop codon readthrough contexts influence reporter expression differentially depending on the presence of an IRES

Background: Previously we reported the discovery of stop codon readthrough in AMD1 mRNA followed by ribosome stalling at the end of a conserved Open Reading Frame (ORF) that we termed AMD1. To explain the severe suppression of reporters fused to AMD1 tail we proposed a mechanism invoking ribosome queueing. To test this hypothesis, we placed the reporter stop codon in the context of readthrough permissive sequences in a dual reporter vector with downstream reporter expression driven by the EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: We employ dual luciferase assays, western blotting and RT-qPCR to explore the effects of test sequences downstream to the reporter stop codon on its expression in dual and monocistronic reporter vectors. Results: With the dual reporter system, the disproportionate reduction of upstream reporter activity is not specific to AMD1 tail and occurs as long as the readthrough stop codon context is present at the end of the reporter’s ORF. In a monocistronic vector without an IRES, the test sequences had distinct effects which were reflective of their properties e.g. AMD1 tail inhibitory effect. We further show with RT-qPCR that the EMCV IRES driven expression of a reporter is an accurate proxy of reporter RNA levels. Conclusions: While our findings provide little new information regarding the functional role of AMD1 tail, they raise caution for the use of viral IRES elements in expression vectors for studying mechanisms of mRNA translation. These findings may also be pertinent to the natural properties of read through permissive sequences and of IRES elements, though these require a separate investigation.

Modular and Molecular Optimization of a LOV (Light–Oxygen–Voltage)-Based Optogenetic Switch in Yeast

Optogenetic switches allow light-controlled gene expression with reversible and spatiotemporal resolution. In Saccharomyces cerevisiae, optogenetic tools hold great potential for a variety of metabolic engineering and biotechnology applications. In this work, we report on the modular optimization of the fungal light–oxygen–voltage (FUN-LOV) system, an optogenetic switch based on photoreceptors from the fungus Neurospora crassa. We also describe new switch variants obtained by replacing the Gal4 DNA-binding domain (DBD) of FUN-LOV with nine different DBDs from yeast transcription factors of the zinc cluster family. Among the tested modules, the variant carrying the Hap1p DBD, which we call “HAP-LOV”, displayed higher levels of luciferase expression upon induction compared to FUN-LOV. Further, the combination of the Hap1p DBD with either p65 or VP16 activation domains also resulted in higher levels of reporter expression compared to the original switch. Finally, we assessed the effects of the plasmid copy number and promoter strength controlling the expression of the FUN-LOV and HAP-LOV components, and observed that when low-copy plasmids and strong promoters were used, a stronger response was achieved in both systems. Altogether, we describe a new set of blue-light optogenetic switches carrying different protein modules, which expands the available suite of optogenetic tools in yeast and can additionally be applied to other systems.

CG dinucleotide removal in bioluminescent and fluorescent reporters improves HIV-1 replication and reporter gene expression for dual imaging in humanized mice

Visualizing the transmission and dissemination of human immunodeficiency virus type 1 (HIV-1) in real time in humanized mouse models is a robust tool to investigate viral replication during treatments and in tissue reservoirs. However, the stability and expression of HIV-1 reporter genes are obstacles for long-term serial imaging in vivo . Two replication-competent CCR5-tropic HIV-1 reporter constructs were created that encode either nanoluciferase (nLuc) or a near - infrared fluorescent protein (iRFP) upstream of nef . HIV-1 reporter virus replication and reporter gene expression was measured in cell culture and in humanized mice. While reporter gene expression in vivo correlated initially with plasma viremia, expression decreased after 4-5 weeks despite high plasma viremia. The reporter genes were codon-optimized to remove cytosine/guanine (CG) dinucleotides and new CO-nLuc and CO-iRFP viruses were reconstructed. Removal of CG dinucleotides in HIV-1 reporter viruses improved replication in vitro and reporter expression in vivo and ex vivo . Both codon optimized reporter viruses could be visualized during co-infection and in vivo reporter gene expression during treatment failure preceded detection of plasma viremia. While the dynamic range of CO-iRFP HIV-1 was lower than that of CO-nLuc HIV-1, both viruses could have utility in studying and visualizing HIV-1 infection in humanized mice. Importance Animal models are important for studying HIV-1 pathogenesis and treatments. We developed two viruses each encoding a reporter gene that can be expressed in cells after infection. This study shows that HIV-1 infection can be visualized by noninvasive, whole body imaging in mice with human immune cells over time by reporter expression. We improved reporter expression to reflect HIV-1 replication and showed that two viral variants can be tracked over time in the same animal and can predict failure of antiretroviral therapy to suppress virus.

In Vivo Function of Flow-Responsive Cis -DNA Elements of the Endothelial Nitric Oxide Synthase Gene: A Role for Chromatin-Based Mechanisms

Background: Endothelial nitric oxide synthase (eNOS) is an endothelial cell (EC)-specific gene predominantly expressed in medium- to large-sized arteries where ECs experience atheroprotective laminar flow with high shear stress (SS). Disturbed flow with lower average SS decreases eNOS transcription, which leads to the development of atherosclerosis, especially at bifurcations and curvatures of arteries. This prototypic arterial EC gene contains two distinct flow-responsive cis -DNA elements in the promoter, the Shear Stress Response Element (SSRE) and the Krüppel-Like Factor (KLF) element. Previous in vitro studies suggested their positive regulatory functions on flow-induced transcription of EC genes including eNOS. However, the in vivo function of these cis -DNA elements remains unknown. Methods: Insertional transgenic mice with a mutation at each flow-responsive cis-DNA element were generated using a murine eNOS promoter-β-galactosidase reporter by linker-scanning mutagenesis and compared with episomal-based mutations in vitro . DNA methylation at the eNOS proximal promoter in mouse ECs was assessed by bisulfite sequencing or pyrosequencing. Results: Wildtype mice with a functional eNOS promoter-reporter transgene exhibited reduced endothelial reporter expression in the atheroprone regions of disturbed flow (n=5). Surprisingly, the SSRE mutation abrogated reporter expression in ECs and was associated with aberrant hypermethylation at the eNOS proximal promoter (n=7). Reporter gene silencing was independent of transgene copy number and integration position, indicating that the SSRE is a critical cis -element necessary for eNOS transcription in vivo . The KLF mutation demonstrated an integration site-specific decrease in eNOS transcription, again with marked promoter methylation (n=8), suggesting that the SSRE alone is not sufficient for eNOS transcription in vivo. In wildtype mice, the native eNOS promoter was significantly hypermethylated in ECs from the atheroprone regions where eNOS expression was markedly repressed by chronic disturbed flow, demonstrating that eNOS expression is regulated by flow-dependent DNA methylation that is region-specific in the arterial endothelium in vivo . Conclusions: We report, for the first time, that the SSRE and KLF elements are critical flow sensors necessary for a transcriptionally permissive, hypomethylated eNOS promoter in ECs under chronic SS in vivo . Moreover, eNOS expression is regulated by flow-dependent epigenetic mechanisms, which offers novel mechanistic insight on eNOS gene regulation in atherogenesis.

Heterologous reporter expression in the planarian Schmidtea mediterranea through somatic mRNA transfection

Planarians have long been studied for their regenerative abilities, but they possess limited genetic tools due to challenges in gene delivery, expression, and detection, despite decades of work. We developed a toolbox for heterologous protein expression in planarian cells and in live animals. Specifically, we identified and optimized nanotechnological and chemical transfection methods to efficiently deliver mRNA encoding nanoluciferase into somatic cells, including planarian adult stem cells (neoblasts). The use of a luminescent reporter allowed us to quantitatively measure protein expression through spectroscopy and microscopy, thus overcoming the strong autofluorescent background of planarian tissues. Using this platform, we investigated the use of endogenous untranslated region (UTR) sequences and codon usage bias to post-transcriptionally alter gene expression. Our work provides a strong foundation for advancing exogenous gene expression and for the rapid prototyping of genetic constructs to accelerate the development of transgenic techniques in planarians.

SARS-CoV-2 ORF6 Disrupts Bidirectional Nucleocytoplasmic Transport through Interactions with Rae1 and Nup98

ABSTRACT RNA viruses that replicate in the cytoplasm often disrupt nucleocytoplasmic transport to preferentially translate their own transcripts and prevent host antiviral responses. The Sarbecovirus accessory protein ORF6 has previously been shown to be a major inhibitor of interferon production in both severe acute respiratory syndrome coronavirus (SARS-CoV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Here, we show SARS-CoV-2-infected cells display an elevated level of nuclear mRNA accumulation compared to mock-infected cells. We demonstrate that ORF6 is responsible for this nuclear imprisonment of host mRNA, and using a cotransfected reporter assay, we show this nuclear retention of mRNA blocks expression of newly transcribed mRNAs. ORF6’s nuclear entrapment of host mRNA is associated with its ability to copurify with the mRNA export factors, Rae1 and Nup98. These protein-protein interactions map to the C terminus of ORF6 and can be abolished by a single amino acid mutation in Met58. Overexpression of Rae1 restores reporter expression in the presence of SARS-CoV-2 ORF6. SARS-CoV ORF6 also interacts with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly copurifies with Rae1 and Nup98 and results in significantly reduced expression of reporter proteins compared to SARS-CoV ORF6, a potential mechanism for the delayed symptom onset and presymptomatic transmission uniquely associated with the SARS-CoV-2 pandemic. We also show that both SARS-CoV and SARS-CoV-2 ORF6 block nuclear import of a broad range of host proteins. Together, these data support a model in which ORF6 clogs the nuclear pore through its interactions with Rae1 and Nup98 to prevent both nuclear import and export, rendering host cells incapable of responding to SARS-CoV-2 infection. IMPORTANCE SARS-CoV-2, the causative agent of coronavirus disease 2019 (COVID-19), is an RNA virus with a large genome that encodes multiple accessory proteins. While these accessory proteins are not required for growth in vitro, they can contribute to the pathogenicity of the virus. We demonstrate that SARS-CoV-2-infected cells accumulate poly(A) mRNA in the nucleus, which is attributed to the accessory protein ORF6. Nuclear entrapment of mRNA and reduced expression of newly transcribed reporter proteins are associated with ORF6’s interactions with the mRNA export proteins Rae1 and Nup98. SARS-CoV ORF6 also shows the same interactions with Rae1 and Nup98. However, SARS-CoV-2 ORF6 more strongly represses reporter expression and copurifies with Rae1 and Nup98 compared to SARS-CoV ORF6. Both SARS-CoV ORF6 and SARS-CoV-2 ORF6 block nuclear import of a wide range of host factors through interactions with Rae1 and Nup98. Together, our results suggest ORF6’s disruption of nucleocytoplasmic transport prevents infected cells from responding to the invading virus.

Differential RA responsiveness among subsets of mouse late progenitor spermatogonia

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII-VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT-) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X-XII), while Dppa3- late progenitors remained abundant, suggesting that Dppa3+ and Dppa3- late progenitors differentially responded to RA. Following acute RA treatment (2-4hr), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI-IX), than Dppa3- late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.