Dual Reporter Vector Research Articles

The non-canonically organized members of MIR395 gene family in Brassica juncea are associated with developmentally regulated, sulfate-stress responsive bidirectional promoters that exhibit orientation-dependent differential transcriptional activity

Several MICRORNA genes belonging to same family or different families are often found in homologous or non-homologous clusters. Among the various classes, head-to-head arranged genes form one of the largest categories of non-canonically organized genes. Such head-to-head arranged, non-canonically organized genes possibly share cis-regulatory region with the intergenic sequence having the potential to function as bi-directional promoter (BDP). The transcriptional regulation of head-to-head arranged genes, especially with bidirectional promoters, remains an enigma. In the past, bidirectional promoters have been characterized for a small set of protein-coding gene pairs in plants; however, to the best of our knowledge, no such study has been carried so far for MICRORNA genes. The present study thus functionally characterizes bidirectional promoters associated with members of MIR395 family, which is evolutionary conserved and is most frequently occurring cluster across plant kingdom.In Arabidopsis thaliana, the MIR395 gene family contains six members with two head-to-head arranged gene pairs- MIR395A-B and MIR395E-F. This organization was found to be conserved at seven loci for MIR395A-B, and eleven loci for MIR395E-F in five Brassica sps. Sequence analysis of the putative bidirectional promoters revealed variation in length, GC content and distribution of strict TATA-box. Comparatively higher level of conservation at both the ends of the bidirectional promoters, corresponding to ca. 250 bp upstream of 5’end of the respective MIRNA precursor, was observed. These conserved regions harbour several abiotic stress (nutrient, salt, drought) and hormone (ABA, ethylene) responsive cis-motifs. Functional characterization of putative bidirectional promoters associated with MIR395A-B and MIR395E-F from Arabidopsis and their respective orthologs from Brassica juncea (Bj_A08 MIR395A-B, Bj_B03 MIR395A-B, Bj_A07.1 MIR395E-F and Bj_A07.2 MIR395E-F) was carried out using a dual-reporter vector with β-glucuronidase (GUS) and Green Fluorescent Protein (GFP). Analysis of transcriptional regulation of the two reporter genes - GUS and GFP during developmental stages confirmed their bidirectional nature. Orientation-dependent differential reporter activity indicated asymmetric nature of the promoters. Comparison of the reporter activity amongst orthologs, paralogs and homeologs revealed regulatory diversification, an outcome expected in polyploid genomes. Interestingly, reporter gene activities driven by selected bidirectional promoters were also observed in anther and siliques apart vegetative tissues indicating role of miR395 in anther and fruit development. Finally, we evaluated the activity of reporter genes driven under transcriptional regulation of bidirectional promoters under normal and sulfate-deprived conditions which revealed asymmetric inducibility under sulfate-starvation, in agreement with the known role of miR395 in sulfate homeostasis.

Development of dual reporter vector system for estimating translational activity of regulatory elements

BackgroundFor the needs of modern biotechnology, a quantitative approach to the control of regulatory elements at all stages of gene expression has long become indispensable. Such a control regime is impossible without a quantitative analysis of the role of each regulatory element or pattern used. Therefore, it seems important to modify and develop the accuracy, reproducibility, and availability of methods for quantifying the contribution of each regulatory code to the implementation of genetic information.ResultsA new vector system for transient expression in plants is described; this system is intended for quantitative analysis of the contribution of regulatory elements to transcription and translation efficiencies. The proposed vector comprises two expression cassettes carrying reporter genes (of the Clostridium thermocellum thermostable lichenase and E. coliβ-glucuronidase) under the control of different promoters. Herewith we also propose a new method for quantification of the effect of tested regulatory elements on expression, which relies on assessment of the enzyme activities of reporter proteins taking into account the transcription of their genes.ConclusionsIn our view, this approach makes it possible to precisely determine the amounts of reporter proteins and their transcripts at all stages of expression. The efficiency of the proposed system has been validated by the analysis of the roles of known translation enhancers at the stages of transcription and translation.

MiR-128-1-5p regulates differentiation of ovine stromal vascular fraction by targeting the KLF11 5′-UTR

Fat content is an important index to evaluate the individual performance of livestock animals such as sheep for meat production purposes. Reducing the subcutaneous and visceral fat while increasing the intramuscular fat is a valuable goal to achieve for the meat production industry. Here, we investigated the effect of miR-128-1-5p on adipogenesis of subcutaneous fat by targeting 5’-UTR in KLF11, a rare mechanism where most miRNAs bind the 3’-UTR of mRNAs. A dual fluorescence reporter assay was conducted to validate the binding sites of miR-128-1-5p on 5’-UTR of KLF11 mRNA. Roles of miR-128-1-5p in KLF11 expression were measured through co-transfecting miRNA mimics with KLF11-expressing vectors (CDSs together with or without the 5′-UTR) into ovine stromal vascular fractions (SVF). Additionally, functional roles of miR-128-1-5p, and KLF11 in adipogenesis of ovine subcutaneous fat were investigated. Results showed that miR-128-1-5p targeted KLF11 5′-UTR, reduced the fluorescence activity of the dual fluorescent reporter vector, as well as KLF11 mRNA, and protein expression levels. During the differentiation of SVF, disturbing the expression of miR-128-1-5p and KLF11 changed the adipogenic differentiation of SVF as observed in the lipid formation, and adipogenic marker genes. This study indicates that miR-128-1-5p promotes the expression of lipogenic marker genes and the formation of lipid droplets by targeting KLF11 5′-UTR. Furthermore, overexpression, and inhibition of KLF11 indicate that KLF11 inhibited SVF differentiation. In summary, the 5’-UTR binding mechanism discovered in this study extends the understanding of miRNA functions. Key roles of miR-128-1-5p and KLF11 in the adipogenesis of sheep subcutaneous fat have potential values for improving the meat and/or fat ratio of domestic animals.

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. In the original study, we tested this hypothesis, by placing 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:Here 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 by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

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.

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. In the original study, we tested this hypothesis, by placing 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: Here 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 by employing RT-qPCR that in the IRES vectors, the Fluc activity levels measured by the luciferase assay are an accurate proxy of 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

Th17 T Cells and Immature Dendritic Cells Are the Preferential Initial Targets after Rectal Challenge with a Simian Immunodeficiency Virus-Based Replication-Defective Dual-Reporter Vector.

Understanding the earliest events of human immunodeficiency virus (HIV) sexual transmission is critical to developing and optimizing HIV prevention strategies. To gain insights into the earliest steps of HIV rectal transmission, including cellular targets, rhesus macaques were intrarectally challenged with a single-round simian immunodeficiency virus (SIV)-based dual reporter that expresses luciferase and near-infrared fluorescent protein 670 (iRFP670) upon productive transduction. The vector was pseudotyped with the HIV-1 envelope JRFL. Regions of tissue containing foci of luminescent transduced cells were identified macroscopically using an in vivo imaging system, and individual transduced cells expressing fluorescent protein were identified and phenotyped microscopically. This system revealed that anal and rectal tissues are both susceptible to transduction 48 h after the rectal challenge. Detailed phenotypic analysis revealed that, on average, 62% of transduced cells are CCR6-positive (CCR6+) T cells-the vast majority of which express RORγT, a Th17 lineage-specific transcription factor. The second most common target cells were immature dendritic cells at 20%. These two cell types were transduced at rates that are four to five times higher than their relative abundances indicate. Our work demonstrates that Th17 T and immature dendritic cells are preferential initial targets of HIV/SIV rectal transmission. IMPORTANCE Men and women who participate in unprotected receptive anal intercourse are at high risk of acquiring HIV. While in vitro data have developed a framework for understanding HIV cell tropism, the initial target cells in the rectal mucosa have not been identified. In this study, we identify these early host cells by using an innovative rhesus macaque rectal challenge model and methodology, which we previously developed. Thus, by shedding light on these early HIV/SIV transmission events, this study provides a specific cellular target for future prevention strategies.

A high-content drug screening strategy to identify protein level modulators for genetic diseases: A proof-of-principle in autosomal dominant leukodystrophy.

In genetic diseases, the most prevalent mechanism of pathogenicity is an altered expression of dosage-sensitive genes. Drugs that restore physiological levels of these genes should be effective in treating the associated conditions. We developed a screening strategy, based on a bicistronic dual-reporter vector, for identifying compounds that modulate protein levels, and used it in a pharmacological screening approach. To provide a proof-of-principle, we chose autosomal dominant leukodystrophy (ADLD), an ultra-rare adult-onset neurodegenerative disorder caused by lamin B1 (LMNB1) overexpression. We used a stable Chinese hamster ovary(CHO) cell line that simultaneously expresses an AcGFP reporter fused to LMNB1 and a Ds-Red normalizer. Using high-content imaging analysis, we screened a library of 717 biologically active compounds and approved drugs, and identified alvespimycin, an HSP90 inhibitor, as a positive hit. We confirmed that alvespimycin can reduce LMNB1 levels by 30%-80% in five different cell lines (fibroblasts, NIH3T3, CHO, COS-7, and rat primary glial cells). In ADLD fibroblasts, alvespimycin reduced cytoplasmic LMNB1 by about 50%. We propose this approach for effectively identifying potential drugs for treating genetic diseases associated with deletions/duplications and paving the way towardPhase II clinical trials.

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 (AMD1 tail). 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 governed by EMCV IRES. In accordance with our hypothesis, we observed a striking disproportional reduction of upstream reporter activity in response to increased readthrough levels. Methods: Here we employ dual luciferase assay and western blotting to explore the effects of AMD1 tail and control sequences on reporter 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. The decreased reporter activity that appears to be induced by the readthrough sequence occurs only in reporters containing EMCV IRES. Monocistronic reporters with the same readthrough context sequence exhibit only a modest reduction in reporter activity. Furthermore, in monocistronic vectors, the disproportionate reduction of reporter levels greatly increased when AMD1 tail was translated as a result of readthrough. Such readthrough-mediated reduction was not observed when AMD1 tail was substituted with unrelated sequences in agreement with our original hypothesis. 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 readthrough permissive sequences and of IRES elements, though these require a separate investigation.

A series of dual-reporter vectors for ratiometric analysis of protein abundance in plants.

Ratiometric reporter systems enable comparisons of the abundance of a protein of interest, or “target,” relative to a reference protein. Both proteins are encoded on a single transcript but are separated during translation. This arrangement bypasses the potential for discordant expression that can arise when the target and reference proteins are encoded by separate genes. We generated a set of 18 Gateway‐compatible vectors termed pRATIO that combine a variety of promoters, fluorescent, and bioluminescent reporters, and 2A “self‐cleaving” peptides. These constructs are easily modified to produce additional combinations or introduce new reporter proteins. We found that mScarlet‐I provides the best signal‐to‐noise ratio among several fluorescent reporter proteins during transient expression experiments in Nicotiana benthamiana. Firefly and Gaussia luciferase also produce high signal‐to‐noise in N. benthamiana. As proof of concept, we used this system to investigate whether degradation of the receptor KAI2 after karrikin treatment is influenced by its subcellular localization. KAI2 is normally found in the cytoplasm and the nucleus of plant cells. In N. benthamiana, karrikin‐induced degradation of KAI2 was only observed when it was retained in the nucleus. These vectors are tools to easily monitor in vivo the abundance of a protein that is transiently expressed in plants, and will be particularly useful for investigating protein turnover in response to different stimuli.

Chloroplast Dual Divergent Promoter Plasmid for Heterologous Protein Expression in Tetraselmis suecica (Chlorophyceae, Chlorodendrales).

The eukaryotic green microalga Tetraselmis suecica is commonly used for aquaculture purposes because of its high stress tolerance and ease of culture in a wide spectrum of environments; they are therefore suitable candidates for biotechnology applications. To date, no data are available regarding chloroplast transformation vectors based on specific endogenous promoters and homologous targeting regions. We report on the identification of Tetraselmis suecica genes encoding the ribulose bisphosphate carboxylase/oxygenase large subunit protein, the photosystem II D1 protein and the ATP synthase CF1-beta subunit protein together with their untranslated regions (5'UTR, 3'UTR). The full-length ORFs of the putative genes with their regulatory sequences were obtained. We were also able to identify the downstream 3' end of the large subunit ribosomal RNA gene (23S) along with the 5S RNA end-to-end with the psbA gene on the complementary strand. The intergenic region between these genes appears to be a good target site for the integration of target proteins. Moreover, we identified a back-to-back promoter region among the rbcL and atpB genes. To assess the bidirectionality activities of both promoters, a dual reporter vector was constructed for Tetraselmis suecica transformation containing the cat and TurboGFP genes driven by the 5'rbcL/5'atpB divergent promoter. The vector included the 23S-5S and psbA nucleotide sequences as flanking regions. These flanking regions provided suitable insertionsites within the chloroplast genome forcassette integration via homologous recombination.Simultaneous expression of the chloramphenicol-resistant conferring gene and the gene coding for TurboGFP driven by 5'rbcL/5'atpB showed a potent natural bidirectional promoter as a reliable genetic tool.

Serum starvation enhances nonsense mutation readthrough.

Of all genetic mutations causing human disease, premature termination codons (PTCs) that result from splicing defaults, insertions, deletions, and point mutations comprise around 30%. From these mutations, around 11% are a substitution of a single nucleotide that change a codon into a premature termination codon. These types of mutations affect several million patients suffering from a large variety of genetic diseases, ranging from relatively common inheritable cancer syndromes to muscular dystrophy or very rare neuro-metabolic disorders. Over the past three decades, genetic and biochemical studies have revealed that certain antibiotics and other synthetic molecules can act as nonsense mutation readthrough-inducing drugs. These compounds bind a specific site on the rRNA and, as a result, the stop codon is misread and an amino acid (that may or may not differ from the wild-type amino acid) is inserted and translation occurs through the premature termination codon. This strategy has great therapeutic potential. Unfortunately, many readthrough agents are toxic and cannot be administered over the extended period usually required for the chronic treatment of genetic diseases. Furthermore, readthrough compounds only restore protein production in very few disease models and the readthrough levels are usually low, typically achieving no more than 5% of normal protein expression. Efforts have been made over the years to overcome these obstacles so that readthrough treatment can become clinically relevant. Here, we present the creation of a stable cell line system that constitutively expresses our dual-reporter vector harboring two cancer initiating nonsense mutations in the adenomatous polyposis coli (APC) gene. This system will be used as an improved screening method for isolation of new nonsense mutation readthrough inducers. Using these cell lines as well as colorectal cancer cell lines, we demonstrate that serum starvation enhances drug-induced readthrough activity, an observation which may prove beneficial in a therapeutic scenario that requires higher levels of the restored protein. KEY MESSAGES: Nonsense mutations affects millions of people worldwide. We have developed a nonsense mutation read-through screening tool. We find that serum starvation enhances antibiotic-induced nonsense mutation read-through. Our results suggest new strategies for enhancing nonsense mutation read-through that may have positive effects on a large number of patients.

Molecular characterization of ABHD5 gene promoter in intramuscular preadipocytes of Qinchuan cattle: Roles of Evi1 and C/EBPα

The genetic regulation of lipolytic enzyme is closely related to carcass quality traits through deposition of intramuscular fat (marbling) in beef cattle breeds. The α/β hydrolase domain containing 5 (ABHD5) is an accelerating factor of adipose triglyceride lipase (ATGL), which plays a key role in triglyceride metabolism. In this study, we determined that bovine ABHD5 gene was highly expressed in adult bovine adipose tissue. To elucidate the molecular mechanisms involved in bovine ABHD5 regulation, we cloned and characterized the promoter region of ABHD5. Applying 5′-rapid amplification of cDNA end analysis (RACE), we identified transcriptional start site (TSS) found in the predicted CpG island within promoter region of ABHD5 gene. Using the recombinant dual fluorescent reporter vectors, the fragment of –109/+307 was identified as proximal minimum core promoter region of ABHD5 in bovine intramuscular adipocytes. Site directed mutagenesis and electrophoretic mobility shift assay (EMSA) confirmed the role of two transcription factors, namely Ectopic viral integration site-1 (Evi1) and CCAAT/enhancer binding protein alpha (C/EBPα), in the regulation of ABHD5 gene. Taken together these findings we can conclude that ABHD5 gene regulated by Evi1 and C/EBPα could be used as potential marker in marker assisted selection for the improvement of Qinchuan cattle breed for carcass quality traits.

Aminoglycoside-stimulated readthrough of premature termination codons in selected genes involved in primary ciliary dyskinesia

ABSTRACTTranslational readthrough of premature termination codons (PTCs) induced by pharmacological compounds has proven to be an effective way of restoring functional protein expression and reducing symptoms in several genetic disorders. We tested the potential of different concentrations of several aminoglycosides (AAGs) for promoting PTC-readthrough in 5 genes involved in the pathogenesis of primary ciliary dyskinesia, an inherited disorder caused by the dysfunction of motile cilia and flagella. The efficiency of readthrough stimulation of PTCs cloned in dual reporter vectors was examined in 2 experimental settings: in vitro (transcription/translation system) and ex vivo (transiently transfected epithelial cell line). PTC-readthrough was observed in 5 of the 16 mutations analyzed. UGA codons were more susceptible to AAG-stimulated readthrough than UAG; no suppression of UAA was observed. The efficiency of PTC-readthrough in vitro (from less than 1% to ∼28% of the translation from the corresponding wild-type constructs) differed with the AAG type and concentration, and depended on the combination of AAG and PTC, indicating that each PTC has to be individually tested with a range of stimulating compounds. The maximal values of PTC suppression observed in the ex vivo experiments were, depending on AAG used, 3–5 times lower than the corresponding values in vitro, despite using AAG concentrations that were 2 orders of magnitude higher. This indicates that, while the in vitro system is sufficient to examine the readthrough-susceptibility of PTCs, it is not sufficient to test the compounds potential to stimulate PTC-readthrough in the living cells. Most of the tested compounds (except for G418) at their highest concentrations did not disturb ciliogenesis in the cultures of primary respiratory epithelial cells from healthy donors.

Isolation and Functional Characterization of Bidirectional Promoters in Rice.

Bidirectional promoters, which show great application potential in genetic improvement of plants, have aroused great research interest recently. However, most bidirectional promoters were cloned individually in the studies of single genes. Here, we initiatively combined RNA-seq data and cDNA microarray data to discover the potential bidirectional promoters in rice genome. Based on the expression level and correlation of each adjacent and oppositely transcribed gene pair, we selected four candidate gene pairs. Then, the intergenic region between each pair was isolated and cloned into a dual reporter vector pDX2181 for functional identification. GUS and GFP assays of the transgenic plants indicated that all the intergenic regions showed bidirectional expression activity in various tissues. Through 5′ and 3′ deletion analysis on one of the above bidirectional promoters, we identified the enhancing region which sharply increased its bidirectional expression efficiency and the essential regions respectively responsible for its 5′ and 3′ basic expression activity. The bidirectional arrangement of the four gene pairs in six gramineous plants was also analyzed, showing the conserved characteristics of the four bidirectional promoters identified in our study. In addition, two novel cis-sequences conserved in the four bidirectional promoters were discovered by bioinformatic identification. Our study proposes a feasible method for selecting, cloning, and functionally identifying bidirectional promoters as well as for discovering their bidirectional regulatory regions and conserved sequences in rice.

Rapid Determination of HIV-1 Mutant Frequencies and Mutation Spectra Using an mCherry/EGFP Dual-Reporter Viral Vector.

The high mutation rate of human immunodeficiency virus type-1 (HIV-1) has been a pivotal factor in its evolutionary success as a human pathogen, driving the emergence of drug resistance, immune system escape, and invasion of distinct anatomical compartments. Extensive research has focused on understanding how various cellular and viral factors alter the rates and types of mutations produced during viral replication. Here, we describe a single-cycle dual-reporter vector assay that relies upon the detection of mutations that eliminate either expression of mCherry or enhanced green fluorescent protein (EGFP). The reporter-based method can be used to efficiently quantify changes in mutant frequencies and mutation spectra that arise due to a variety of factors, including viral mutagens, drug resistance mutations, cellular physiology, and APOBEC3 proteins.

Abstract 5110: Initial characterization of an optical reporter myoblast cell line for non-invasive imaging in a cancer cachexia model in mice

Abstract Cancer associated cachexia affects a majority of patients during cancer progression, compromising therapeutic interventions and contributing to decreased survival rates. Identifying factors involved in the onset of cachexia will provide a better understanding of early treatment strategies. To this end, we developed a mouse model system that allows for real time longitudinal monitoring of cancer induced wasting and has the potential of identifying early cachectic events. Several attributes of our system are new. 1) The construction of a dual optical reporter vector with green fluorescence protein (GFP) expression constitutively driven from an EF1α promoter and red fluorescence protein (tdTomato) expression driven by an engineered skeletal muscle specific inducible promoter. The latter is a synthetic sequence of a triple-tandem repeat of the glucocorticoid-FOXO1 response element region from the proximal promoter of the human MuRF1 gene. 2) Generation of a rat L6 myoblast optical reporter cell line (To3B cells) with stable integration of the dual reporter vector construct, which provides living reporter grafts within mouse muscle. 3) A human pancreatic cancer cell line (Pa04C) that as an orthotopic or subcutaneous xenograft causes weight loss in male SCID mice. In preliminary studies, we tested several human pancreatic cancer cell lines as orthotopic xenografts in male SCID mice. We found that red fluorescence signals were reproducibly detected in live mice only from To3B grafts in mice undergoing weight loss, while graft size and viability were readily monitored by imaging GFP fluorescence in all animals. In addition, mice bearing Pa04C tumors lost the most weight while mice bearing Panc1 tumors gained weight. Therefore, Pa04C and Panc1 cells were used for subcutaneous xenografts in male SCID mice and weight loss was followed with optical monitoring of To3B grafts. Importantly, in weight losing mice, we found that red fluorescence could be detected and quantified at a nascent stage of the syndrome; e.g., unambiguous red fluorescent signals were quantified at weight losses of only 1.2 to 2.7% at very low tumor burdens of only ∼0.079 to ∼0.170 cm3. Red fluorescence remained very low to undetectable in mice that gained weight. Tumor sizes were comparable between groups, which was an indication that factors independent of tumor growth were involved in switching on red fluorescence. Ex vivo fluorescence microscopy confirmed a robust presence of red fluorescence only in To3B grafts in skeletal muscle from Pa04C tumor bearing mice. The evidence from this initial development of a unique optical reporter myoblast cell line indicates the potential to detect the onset of cancer cachexia. These studies set the ground work for future research aimed at identifying initiating systemic as well as local molecular events in the muscle of cachectic mice. Supported by NIH P50CA103175 Citation Format: Paul T. Winnard, Marie-France Penet, Yelena Mironchik, Flonne Wildes, Anirban Maitra, Zaver M. Bhujwalla. Initial characterization of an optical reporter myoblast cell line for non-invasive imaging in a cancer cachexia model in mice. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5110. doi:10.1158/1538-7445.AM2015-5110

Construction and validation of a novel dual reporter vector for studying mammalian bidirectional promoters

Regulation of gene expression plays important role in cellular functions. With the development of sequencing techniques, more and more genomes are available and genome-wide analyses of genomic structures that may affect gene expression regulation are now possible. Analyses of several genomes have found a class of regulatory regions that contain elements that initiate transcription of two different genes positioned with a head-to-head arrangement in two opposite directions. These regulatory regions are known as bidirectional promoters. Although bidirectional promoters have been known for years, recent genome-scale studies have shown that the regulation of the expression of up to 10% of the genes are controlled by bidirectional promoters. These findings are based mostly on computational work and only a limited number of putative bidirectional promoters have been experimentally validated. Developing methods to study bidirectional promoters will allow researchers to understand how these regions are regulated and the roles that divergent transcription plays in the expression of genes. Here, we have developed a novel dual-fluorescence reporter gene vector to study the transcriptional output of mammalian bidirectional promoters. We demonstrate that this vector is capable of expressing reporter genes under the control of bidirectional promoters, using the known human OSGEP/APEX bidirectional promoter.

Systemic delivery of a breast cancer-detecting adenovirus using targeted microbubbles

One of the major limitations of cancer gene therapy using recombinant human adenovirus (Ad) is rapid Ad inactivation from systemic delivery. To eliminate this, biotin-coated ultrasound contrast agents, or microbubbles (MBs), were streptavidin-coupled with biotinylated antibodies to three distinct tumor vasculature-associated receptors (α(V)β(3) integrin, P-selectin and vascular endothelial growth factor receptor-2) for systemic targeting of a previously generated vector Ad5/3-Id1-SEAP-Id1-mCherry. This cancer-specific, dual-reporter vector was loaded in the targeted MBs and confirmed by confocal microscopy. MB loading capacity was estimated by functional assays as 4.72 ± 0.2 plaque forming unit (PFU) per MB. Non-loaded (free) Ad particles were effectively inactivated by treatment with human complement. The Ad-loaded, targeted-MBs were injected systemically in mice bearing MDA-MB-231 tumors (Grp 1) and compared with two control groups: Ad-loaded, non-targeted MBs (Grp 2) and free Ad (Grp 3) administered under the same conditions. Two days after administration the blood levels of secreted embryonic alkaline phosphatase (SEAP) reporter in Grp 1 mice (16.1 ng ml(-1) ± 2.5) were significantly higher (P<0.05) than those in Grp 2 (9.75 ng ml(-1) ± 1.5) or Grp 3 (4.26 ng ml(-1) ± 2.5) animals. The targeted Ad delivery was also confirmed by fluorescence imaging. Thus, Ad delivery by targeted MBs holds potential as a safe and effective system for systemic Ad delivery for the purpose of cancer screening.

Generation of Sperms Containing EGFP-LacZ Following Transfection of Chicken Testis with a Eukaryotic Dual Reporter Vector

Male germ cells modified by foreign genes can be used to generate transgenic chicken. In this study, in vivo transfection of chicken testis with an EGFP-LacZ dual reporter expression vector was performed. Large-scale plasmid DNA preparation of the EGFP-LacZ eukaryotic expression vector was carried out and efficient transfection of chicken testicle cells using the prepared plasmid DNA was confirmed in vitro. The reporter plasmid was directly injected into adult rooster testes. Semen samples were collected on 10-days post-transfection and every other day thereafter; and a total of six collections were made. Semen slides were subjected to fluorescence microscopy and β-galactosidase activity assay to identify sperms carrying the reporter genes. The presence of EGFP and LacZ was further confirmed by PCR amplification with sperm genomic DNA as template. The testicles of those birds were subjected to cryostat sectioning, fluorescence microscopy and β-galactosidase activity assay. The results showed that sperms with green fluorescence were not observed on semen slides; however, sperms positive for β-galactosidase were detected. Specific amplicons of EGFP and lacZ were detected in four of the six sequentially collected semen samples. Fluorescence microscopy of the corresponding semen slides revealed yellow-green fluorescence, but not clear green fluorescence. The β-galactosidase activity assay and GFP histochemistry using monoclonal antibodies demonstrated positive staining for subsets of testicle cells. Together, these results showed that direct injection of the dual reporter vector into adult rooster testis allowed in vivo transfection of chicken sperm precursor cells, which further developed into sperm containing EGFP-LacZ.