EGFP Reporter Gene Research Articles

IMMU-15. A UNIQUE SELF-STIMULATIVE PROPERTY OF CD70 CAR ADVANCES T CELL FUNCTION AND ANTI-TUMOR RESPONSE IN GLIOBLASTOMA

Abstract BACKGROUND Glioblastoma (GBM) is a refractory brain tumor that desperately needs new therapeutic interventions. Our group identified CD70 as a novel target of CAR-T therapy for this malignancy. We demonstrate that CD70 is overexpressed by low-/high-grade gliomas and associated with poor survival for patients; CD70 promotes CD8 specific cell death and tumor-associated macrophage infiltration in gliomas. The CD70 CAR (using CD27, a natural costimulatory receptor of T cells as an antigen-binding region) T cells can efficiently eradicate CD70 positive tumors in syngeneic and xenograft mouse models. OBJECTIVE To evaluate the properties of CD70 CAR-transduced T cells in GBM treatment. METHODS CD70 CAR or IL13Rα2 CAR was linked with fluorescent reporter gene EGFP, and cloned into a retroviral vector (pMSGV8). In vitro T cell culture and flow cytometry were used to evaluate the self-enrichment property and susceptibility to TCR stimulation of the CAR T cells. KI67, Bcl-2, CD70 gene expression was tested by qPCR to measure the proliferation/apoptosis properties of the CAR T cells. Cytokine profile was analyzed by ELISA. The anti-tumor response was evaluated using Xenograft mouse models. RESULTS Compared with IL13Rα2 CAR T cells, the frequency of CD70 CAR T cells was significantly increased 3 weeks post transduction, and approximately 100 to 150-fold CD70 CAR T cell expansion without additional stimuli was achieved in vitro. The expanded CD70 CAR T cells were mostly (up to 85%) CD8+ T cells three weeks post CAR transduction. Enhanced proliferative capacity and production of IL-2, IFN-γ, and TNF-α of the CD70 CAR-transduced T cells upon anti-CD3/CD28 stimulation were also revealed. Results from animal models show that the CD70 CAR T cells present superior in vivo persistence and antitumor efficacy. CONCLUSION We show the auto-stimulative property, as well as superior T cell function and antitumor efficacy of CD70 CAR T cells in GBM models.

The in vivo detection and measurement of the unfolded protein response in recombinant cellulase producing Saccharomyces cerevisiae strains.

The yeast Saccharomyces cerevisiae possesses industrially desirable traits for ethanol production and has been engineered for consolidated bioprocessing (CBP) of lignocellulosic biomass through heterologous cellulase expression. However, S. cerevisiae produces low titers of cellulases and one suspected reason for this is that heterologous proteins induce the unfolded protein response (UPR). Current methods of measuring the UPR are RNA based and can be inconsistent and cumbersome. We developed vector-based biosensors that will detect and quantify UPR activation. The vector consisted of either the Trichoderma reesei xylanase 2 or codon optimized green fluorescent protein (eGFP) reporter genes under the control of the S. cerevisiae PHAC1 or PKAR2 promoters. The eGFP reporter under control of PKAR2 was identified as the preferred combination due to its superior dynamic range and its greater sensitivity when measuring UPR induction in cellulase producing strains. To our knowledge, we show for the first time that significant UPR activation differences could consistently be observed for different cellulase candidate genes unlike previous RNA-based tests, which were unable to detect these differences. The ability to quantify UPR induction will assist in identifying candidate cellulase genes that do not greatly induce the UPR, making them favorable for use in CBP yeasts.

Efficient Biolistic Transformation of Immature Citrus Rootstocks Using Phosphomannose-isomerase Selection.

This research utilized the E. coli manA gene encoding phosphomannose isomerase (PMI) selection on sucrose/mannose medium to increase transformation efficiencies after biolistic transformation of two immature citrus rootstock cultivars. Plasmid DNA, containing the manA gene and the enhanced green fluorescent protein (egfp) reporter gene, was bombarded into epicotyl explants of immature Carrizo citrange and Swingle citrumelo. GFP positive shoots were micro-grafted onto in vitro grown immature Carrizo rootstocks. Nineteen transgenic Carrizo shoots were obtained from ten paired shots, and eight Swingle shoots from five paired shots. The mean transformation efficiency of Carrizo was 1.9 transgenics/paired shot while the transformation efficiency of Swingle was comparable at 1.6 transgenics/paired shot. The transformants were analyzed by PCR for the presence of transgenes. Southern blot analysis of eight representative Carrizo transgenic events and four Swingle transgenic events showed that all transgenics had one to three copies of the manA gene. The PMI enzyme activity in the transgenic lines was confirmed using the chlorophenol red assay.

Transcriptional Enhancers in the FOXP2 Locus Underwent Accelerated Evolution in the Human Lineage.

Unique human features, such as complex language, are the result of molecular evolutionary changes that modified developmental programs of our brain. The human-specific evolution of the forkhead box P2 (FOXP2) gene-coding region has been linked to the emergence of speech and language in the human kind. However, little is known about how the expression of FOXP2 is regulated and whether its regulatory machinery evolved in a lineage-specific manner in humans. In order to identify FOXP2 regulatory regions containing human-specific changes, we used databases of human-accelerated noncoding sequences or HARs. We found that the topologically associating domain determined using developing human cerebral cortex containing the FOXP2 locus includes two clusters of 12 HARs, placing the locus occupied by FOXP2 among the top regions showing fast acceleration rates in noncoding regions in the human genome. Using in vivo enhancer assays in zebrafish, we found that at least five FOXP2-HARs behave as transcriptional enhancers throughout different developmental stages. In addition, we found that at least two FOXP2-HARs direct the expression of the reporter gene EGFP to foxP2-expressing regions and cells. Moreover, we uncovered two FOXP2-HARs showing reporter expression gain of function in the nervous system when compared with the chimpanzee ortholog sequences. Our results indicate that regulatory sequences in the FOXP2 locus underwent a human-specific evolutionary process suggesting that the transcriptional machinery controlling this gene could have also evolved differentially in the human lineage.

Cationic Derivatives of Polyisoprenoids and Commercial Lipid‐Based DNA Carriers for Transfection in vivo in Spontaneously Hypertensive Rats

Development of efficient vectors for transfection constitutes a major challenge in modern gene therapy. Viral vectors are considered more effective, but plagued by safety concerns. Research shows that derivatives of polyisoprenoid alcohols (trimethylpolyprenylammonium iodides, called PTAI), may serve as lipofectants. In previous research PTAI were proved to be effective as DNA carriers in vitro and in vivo as carriers of DNA vaccines against influenza virus.In the present study the effectiveness of two compositions (PTAI‐6–8 and PTAI‐10–14, containing a mixture of polyprenols with chain lengths from 6 to 8 subunits and from 10 to 14 subunits, respectively and helper lipids: DOPE (1,2‐dioleoyl‐sn‐glycero‐3‐phosphatidylethanolamine) and DC‐cholesterol (3β‐[N‐(N′,N′‐dimethylaminoethane)‐carbamoyl]cholesterol hydrochloride) was investigated for DNA delivery into rats' cells and compared to the commercially available reagents for transfection: in vivo‐jetPEI® (Polyplus‐transfection) and Avalanche®‐in vivo Transfection Reagent (EZ Biosystems LLC). Selected mixtures were used to enhance the expression of vascular endothelial growth factor type A (VEGF‐A) in spontaneously hypertensive rats (SHR). VEGF deficiency is considered one of the factors in the development of hypertension and related kidney diseases.In preliminary experiments, the mixtures containing lipid‐carriers and plasmid DNA (encoding reporter gene and VEGF‐A) were administered intravenously and observed for 24, 48 or 72 h, to establish the optimal conditions for transfection. Subsequently rats were anesthetized, perfused with PBS, and tissues (liver, spleen, kidney, lung) were collected for analysis. Transfection efficiency was evaluated by flow cytometry (BD FACSCanto™ II system) in single cell suspensions prepared from collected tissues.In almost all tested cell suspensions the level of fluorescence of reporter gene (eGFP or mCherry) was undetectable. Only in the liver higher expression of mCherry was observed with commercial reagents (the % of mCherry positive liver cells with Avalanche® was 42 versus 12% in control group receiving 5% glucose; p<0.05). Due to low efficiency of intravenously applied carriers, we tested if selected mixtures are able to provide stable expression of VEGF‐A gene after direct administration to the kidney. SHR were implanted with osmotic pumps (model 2001, ALZET®) connected to catheters allowing the infusion into the renal medulla.The fluorescence of mCherry in the kidneys was undetectable and there were no changes in tissue, plasma and urine levels of VEGF‐A (ELISA). Unfortunately we were not able to prove the usefulness of PTAI for transfection, however all selected mixtures, including commercially available reagents failed to deliver DNA to renal cellsSupport or Funding InformationNCN 2015/17/N/NZ4/02831This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

The Rapid Methylation of T-DNAs Upon Agrobacterium Inoculation in Plant Leaves.

Agrobacterium tumefaciens has been foundational in the development of transgenic plants for both agricultural biotechnology and plant molecular research. However, the transformation efficiency and level of transgene expression obtained for any given construct can be highly variable. These inefficiencies often require screening of many lines to find one with consistent and heritable transgene expression. Transcriptional gene silencing is known to affect transgene expression, and is associated with DNA methylation, especially of cytosines in symmetric CG and CHG contexts. While the specificity, heritability and silencing-associated effects of DNA methylation of transgene sequences have been analyzed in many stably transformed plants, the methylation status of transgene sequences in the T-DNA during the transformation process has not been well-studied. Here we used agro-infiltration of the eGFP reporter gene in Nicotiana benthamiana leaves driven by either an AtEF1α-A4 or a CaMV-35S promoter to study early T-DNA methylation patterns of these promoter sequences. The T-DNA was examined by amplicon sequencing following sodium bisulfite treatment using three different sequencing platforms: Sanger sequencing, Ion Torrent PGM, and the Illumina MiSeq. Rapid DNA methylation was detectable in each promoter region just 2–3 days post-infiltration and the levels continued to rapidly accumulate over the first week, then steadily up to 21 days later. Cytosines in an asymmetric context (CHH) were the most heavily and rapidly methylated. This suggests that early T-DNA methylation may be important in determining the epigenetic and transcriptional fate of integrated transgenes. The Illumina MiSeq platform was the most sensitive and robust way of detecting and following the methylation profiles of the T-DNA promoters. The utility of the methods was then used to show a subtle but significant difference in promoter methylation during intron-mediated enhancement. In addition, the method was able to detect an increase in promoter methylation when the eGFP reporter gene was targeted by siRNAs generated by co-infiltration of a hairpin RNAi construct.

Prediction and characterization of promoters and ribosomal binding sites of Zymomonas mobilis in system biology era

BackgroundZymomonas mobilis is a model bacterial ethanologen with many systems biology studies reported. Besides lignocellulosic ethanol production, Z. mobilis has been developed as a platform for biochemical production through metabolic engineering. However, identification and rigorous understanding of the genetic origins of cellular function, especially those based in non-coding region of DNA, such as promoters and ribosomal binding sites (RBSs), are still in its infancy. This knowledge is crucial for the effective application of Z. mobilis to new industrial applications of biotechnology for fuels and chemicals production.ResultsIn this study, we explored the possibility to systematically predict the strength of promoters based on systems biology datasets. The promoter strength was clustered based on the expression values of downstream genes (or proteins) from systems biology studies including microarray, RNA-Seq and proteomics. Candidate promoters with different strengths were selected for further characterization, which include 19 strong, nine medium, and ten weak ones. A dual reporter-gene system was developed which included appropriate reporter genes. These are the opmCherry reporter gene driven by the constitutive PlacUV5 promoter for calibration, and EGFP reporter gene driven by candidate promoters for quantification. This dual reporter-gene system was confirmed using the inducible promoter, Ptet, which was used to determine the strength of these predicted promoters with different strengths. In addition, the dual reporter-gene system was applied to determine four synthetic RBSs with different translation initiation rates based on the prediction from bioinformatics server RBS calculator. Our results showed that the correlations between the prediction and experimental results for the promoter and RBS strength are relatively high, with R2 values more than 0.7 and 0.9, respectively.ConclusionsThis study not only identified and characterized 38 promoters and four RBSs with different strengths for future metabolic engineering in Z. mobilis, but also established a flow cytometry-based dual reporter-gene system to characterize genetic elements including, but not limited to the promoters and RBSs studied in this work. This study also suggested the feasibility of predicting and selecting candidate genetic elements based on omics datasets and bioinformatics tools. Moreover, the dual reporter-gene system developed in this study can be utilized to characterize other genetic elements of Z. mobilis, which can also be applied to other microorganisms.

Intracameral Delivery of AAV to Corneal Endothelium for Expression of Secretory Proteins.

AAV9 drives gene expression in a highly selective manner within the corneal endothelium of mice following intracameral inoculation into the anterior chamber of the eye. In principle, this allows genes encoding protein constituents of the secretome (representing up to 20% of the human proteome) to be delivered directly into the aqueous humor. From here the secreted protein moves with the natural flow of the aqueous humor via a pressure gradient and is directed toward the outflow tissues. Such a delivery can be employed to modulate outflow facility and intraocular pressure through interactions at the trabecular meshwork and Schlemm's canal. We provide a protocol for the delivery of AAV to the corneal endothelium, using a CMV-driven eGFP reporter gene as a marker.

Synthesis and Evaluation of a New Type of Small Molecule Epigenetic Modulator Containing Imidazo[1,2-b][1,2,4]triazole Motif.

Epigenetic modifications such as DNA methylation is important for many cellular processes, such as cell differentiation and cell death. The disorder of epigenetic state is closely related to human diseases, especially cancers. DNA methylation is a well-characterized epigenetic modification which is related to gene silencing and is considered as a repressive epigenetic mark. DNA methylation caused gene repression can be derepressed by chemical agents. Small molecules targeting DNA methyltransferases, histone deacetylases, and other regulatory factors can activate genes silenced by DNA methylation. However, more and more studies have shown that histone deacetylation is not the only downstream event of DNA methylation. Some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Recently, through high-throughput screening using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation, we identified seven hit compounds that did not directly target bulk DNA methylation or histone acetylation. Three of them (LX-3, LX-4, LX-5) were proven to selectively activate the p38 MAPK pathway in multiple cell types. In order to identify the exact cellular targets of these compounds, we turn to work on the SAR study of LX-3 by constructing a structurally diverse chemical library based on the imidazo[1,2-b][1,2,4]triazole core structure via diversity-oriented synthesis. Our work provides a general approach to efficiently access diverse heterocyclic molecules with interesting epigenetic modulation activities.

Evaluating the utility of human glucagon-like peptide 1 receptor gene as a novel radionuclide reporter gene: a promising molecular imaging tool.

Radiolabelled ligands of glucagon-like peptide 1 receptor (GLP-1R) have been used to image the GLP-1R-expressing tissues (e.g., islets and insulinoma). Here, we introduced human glucagon-like peptide 1 receptor (hglp-1r) gene as a novel radionuclide reporter gene to broaden its applications in molecular imaging in vivo. Transient and stable baculoviral vectors (BV) were re-constructed and used to transfer the hglp-1r gene or enhanced green fluorescent protein (egfp) reporter gene into the stem cells or tumor cells. Cell proliferation assay and flow cytometry analysis demonstrated that BV-mediated reporter gene transferring and expression was biosafe and highly efficient. The BV-mediated exogenous hGLP-1R in target cells showed same ligand-receptor binding characteristics compared with its counterpart in insulinoma cells. Furthermore, the ligand-receptor binding assay showed a high affinity (IC50 = 0.3708nM) and robust correlation (R2 = 0.9264) between the fluorescein-tagged or radiolabeled ligand probes and exogenous hGLP-1R in target cells. The target cells transferred with BV-mediated hGLP-1R could be clearly visualized in nude mice by micro-PET, which was capable of the purposes of short-term tracking transplanted stem cells or long-term monitoring tumor formation. Then, the image-based analysis and bio-distribution analysis quantitatively confirmed high target-to-background ratio of hGLP-1R-expressing cells. This study also investigated the endogenous GLP-1R-expressing organs/tissues in nude mice in the hGLP-1R radionuclide reporter gene imaging. Summarily, we evaluated the utility of hglp-1r gene as a novel radionuclide reporter gene, and demonstrated that it was a favorable and promising candidate of molecular imaging tool, which would expand the spectrum of radionuclide reporter gene imaging systems.

The histidine kinase slnCl1 of Colletotrichum lindemuthianum as a pathogenicity factor against Phaseolus vulgaris L

Colletotrichum lindemuthianum, the causal agent of anthracnose, is responsible for significant damage in the common bean (Phaseolus vulgaris L.). Unraveling the genetic mechanisms involved in the plant/pathogen interaction is a powerful approach for devising efficient methods to control this disease. In the present study, we employed the Restriction Enzyme-Mediated Integration (REMI) methodology to identify the gene slnCl1, encoding a histidine kinase protein, as involved in pathogenicity. The mutant strain, MutCl1, generated by REMI, showed an insertion in the slnCl1 gene, deficiency of the production and melanization of appressoria, as well as the absence of pathogenicity on bean leaves when compared with the wild-type strain. The slnCl1 gene encodes a histidine kinase class IV called SlnCl1 showing identity of 97% and 83% with histidine kinases from Colletotrichum orbiculare and Colletotrichum gloesporioides, respectively. RNA interference was used for silencing the histidine kinase gene and confirm slnCl1 as a pathogenicity factor. Furthermore, we identified four major genes involved in the RNA interference-mediated gene silencing in Colletotrichum spp. and demonstrated the functionality of this process in C. lindemuthianum. Silencing of the EGFP reporter gene and slnCl1 were demonstrated using qPCR. This work reports for the first time the isolation and characterization of a HK in C. lindemuthianum and the occurrence of gene silencing mediated by RNA interference in this organism, demonstrating its potential use in the functional characterization of pathogenicity genes.

Visualisation and characterisation of mononuclear phagocytes in the chicken respiratory tract using CSF1R-transgenic chickens

The respiratory tract is a key organ for many avian pathogens as well as a major route for vaccination in the poultry industry. To improve immune responses after vaccination of chickens through increased uptake of vaccines and targeting to antigen presenting cells, a better understanding of the avian respiratory immune system is required. Transgenic MacReporter birds were used expressing a reporter gene (eGFP or mApple) under the control of the CSF1R promoter and enhancer in cells of the mononuclear phagocyte (MNP) lineage to visualize the ontogeny of the lymphoid tissue, macrophages and dendritic cells, in the trachea, lung and air sac of birds from embryonic day 18–63 weeks of age. Small aggregates of CSF1R-transgene+ cells start to form at the openings of the secondary bronchi at 1 week of age, indicative of the early development of the organised bronchus-associated lymphoid tissue. Immunohistochemical staining revealed subpopulations of MNPs in the lung, based on expression of CSF1R-transgene, CD11, TIM4, LAMP1, and MHC II. Specialised epithelial cells or M cells covering the bronchus-associated lymphoid tissue expressed CSF1R-transgene and type II pneumocytes expressed LAMP1 suggesting that these epithelial cells are phagocytic and transcytose antigen. Highly organised lymphoid tissue was seen in trachea from 4 weeks onwards. Throughout the air sacs at all ages, CSF1R-transgene+ cells were scattered and at later stages, CSF1R-transgene+ cells lined capillaries. These results will serve as a base for further functional characterization of macrophages and dendritic cells and their role in respiratory diseases and vaccine responses.

Datura innoxia plants hydroponically-inoculated with Agrobacterium rhizogenes display an enhanced growth and alkaloid metabolism

BackgroundThe production of secondary metabolites through the culture of entire plants is of great interest. Soilless culture, such as hydroponics, enables the control of plant growth and metabolism. Specific environmental conditions must be developed to maximize the productivity of medicinal plants used as efficient natural bioreactors. MethodsThe nutrient solution of newly established hydroponic cultures ofDatura innoxia Mill. were inoculated with Agrobacterium rhizogenes (A.r.) wild strains (TR7, TR107, 11325 or 15834). Growth and the alkaloid contents of roots and aerial parts were analyzed. Axenic cultures were also performed with modified TR7 strains containing the egfp or gus reporter gene. In vitro isolated root cultures enabled the phenological and molecular demonstration of gene transfer. ResultsA.r.TR 7 led to a greater improvement in plant secondary metabolism and growth. Positive expression of the reporter genes occurred. Isolation and subculture of some of the roots of these plants showed a hairy root phenotype; molecular tests proved the transfer of bacterial genes into the roots isolated from the plants. ConclusionsHyoscyamine and scopolamine productivity is enhanced after A.r. inoculation in the nutrient solution of hydroponic plants. Transformation events occur in the original roots of the plants. This leads to chimeric plants with a part of their roots harboring a hairy root phenotype. Such semi-composite plants could be used for successful specialized metabolite bioproduction in greenhouses.

Minor variation in the 3' downstream region of eGFP reporter gene substantially increases level of its expression in mouse and human cells

Minor variation in the 3' downstream region of eGFP reporter gene substantially increases level of its expression in mouse and human cells

CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system has emerged as a powerful tool for knock-in of DNA fragments via donor plasmid and homology-independent DNA repair mechanism; however, conventional integration includes unnecessary plasmid backbone and may result in the unfaithful expression of the modified endogenous genes. Here, we report an efficient and precise CRISPR/Cas9-mediated integration strategy using a donor plasmid that harbors 2 of the same cleavage sites that flank the cassette at both sides. After the delivery of donor plasmid, together with Cas9 mRNA and guide RNA, into cells or fertilized eggs, concurrent cleavages at both sides of the exogenous cassette and the desired chromosomal site result in precise targeted integration without plasmid backbone. We successfully used this approach to precisely integrate the EGFP reporter gene into the myh6 locus or the GAPDH locus in Xenopus tropicalis or human cells, respectively. Furthermore, we demonstrate that replacing conventional terminators with the endogenous 3UTR of target genes in the cassette greatly improves the expression of reporter gene after integration. Our efficient and precise method will be useful for a variety of targeted genome modifications, not only in X. tropicalis, but also in mammalian cells, and can be readily adapted to many other organisms.-Mao, C.-Z., Zheng, L., Zhou, Y.-M., Wu, H.-Y., Xia, J.-B., Liang, C.-Q., Guo, X.-F., Peng, W.-T., Zhao, H., Cai, W.-B., Kim, S.-K., Park, K.-S., Cai, D.-Q., Qi, X.-F. CRISPR/Cas9-mediated efficient and precise targeted integration of donor DNA harboring double cleavage sites in Xenopus tropicalis.

A study on the use of strain-specific and homologous promoters for heterologous expression in industrial Saccharomyces cerevisiae strains

Polymorphism is well known in Saccharomyces cerevisiae strains used for different industrial applications, however little is known about its effects on promoter efficiency. In order to test this, five different promoters derived from an industrial and a laboratory (S288c) strain were used to drive the expression of eGFP reporter gene in both cells. The ADH1 promoter (PADH1) in particular, which showed more polymorphism among the promoters analyzed, also exhibited the highest differences in intracellular fluorescence production. This was further confirmed by Northern blot analysis. The same behavior was also observed when the gene coding for secreted α-amylase from Cryptococcus flavus was placed under the control of either PADH1. These results underline the importance of the careful choice of the source of the promoter to be used in industrial yeast strains for heterologous expression.

MiRNA122a regulation of gene therapy vectors targeting hepatocellular cancer stem cells.

In this study, we report a miRNA122a based targeted gene therapy for hepatocellular cancer stem cells (CSCs). First, we assessed the levels of miRNA122a in normal human hepatocytes, a panel of hepatocellular carcinoma (HCC) cell lines and hepatocellular CSCs observing its significant downregulation in HCC and CSCs. The miRNA122a binding site was then incorporated at the 3’-UTR of reporter genes gaussia luciferase (GLuc) and eGFP which resulted in significant hepatocyte detargeting. Using this strategy for the delivery of gene directed enzyme prodrug therapy (GDEPT) utilizing the cytosine deaminase/5-fluorocytosine (CD/5-FC) system, we showed significant killing in cells with low or no miRNA122a while those cells, such as hepatocytes with high miRNA122a were largely spared. Next, we showed that CSC enriched tumorspheres exhibit a significant downregulation of miRNA122a expression providing a rational to exploit its binding site for targeted gene delivery. Using plasmids harboring reporters GLuc and eGFP with or without miR122a binding sites, we showed high reporter expression in the CSCs and little reported expression in the non-enriched cultures. Finally, we demonstrate the efficacy of miRNA122a based post-transcriptionally targeted GDEPT for hepatocellular CSCs.

Small molecules capable of activating DNA methylation–repressed genes targeted by the p38 mitogen-activated protein kinase pathway

Regulation of gene expression by epigenetic modifications such as DNA methylation is crucial for developmental and disease processes, including cell differentiation and cancer development. Genes repressed by DNA methylation can be derepressed by various compounds that target DNA methyltransferases, histone deacetylases, and other regulatory factors. However, some additional, unknown mechanisms that promote DNA methylation-mediated gene silencing may exist. Chemical agents that can counteract the effects of epigenetic repression that is not regulated by DNA methyltransferases or histone deacetylases therefore may be of research interest. Here, we report the results of a high-throughput screen using a 308,251-member chemical library to identify potent small molecules that derepress an EGFP reporter gene silenced by DNA methylation. Seven hit compounds were identified that did not directly target bulk DNA methylation or histone acetylation. Analyzing the effect of these compounds on endogenous gene expression, we discovered that three of these compounds (compounds LX-3, LX-4, and LX-5) selectively activate the p38 mitogen-activated protein kinase (MAPK) pathway and derepress a subset of endogenous genes repressed by DNA methylation. Selective agonists of the p38 pathway have been lacking, and our study now provides critical compounds for studying this pathway and p38 MAPK-targeted genes repressed by DNA methylation.

Engineering riboswitch in L. major: From prediction to conceptualization

Post-transcriptional gene regulation is a vital process to regulate expression of the key genes in the eukaryotic cell. Such processes are essential for pathogens which reside inside the host cell. One such pathogen is Leishmania major, which causes cutaneous leishmaniasis. The parasite lives inside the macrophages of mammalian host (mostly human). Inside the macrophage, Leishmania genes show complex host-pathogen interaction regulating a plethora of gene expression. Till date, most of the studies have shown this kind of regulation with respect to the host macrophages. Here, based on an extensive in silico analysis, we have hypothesized a novel Theophylline binding riboswitch mediated post-transcriptional regulation of a gene i.e. RNA Polymerase III subunit1 (Lmjf_09_1060), an essential gene for the parasite's survival both in its promastigote as well as in its amastigote form. Later, we have conceptualized the working of the identified putative Theophylline binding riboswitch cassette in in vitro using E. coli based reporter assay, wherein, a reporter gene (eGFP) is used instead of RNA Polymerase III subunit1 gene and apparently have shown the downregulation of the reporter gene (eGFP) expression under the influence of in silico identified Theophylline binding riboswitch.

PiRNA-like small RNAs are responsible for the maternal-specific knockdown in the ascidian Ciona intestinalis Type A

The mRNAs stored in eggs are crucial for embryogenesis. To address functions of maternal mRNAs, we recently reported the novel method MASK (maternal mRNA-specific knockdown), which we used to specifically knockdown maternal transcripts in the ascidian Ciona intestinalis Type A. In MASK, the cis element of a maternal gene is fused with eGFP or Kaede reporter gene, and the cassette is introduced into Ciona genome by transposon-mediated transgenesis. In eggs of the transgenic lines, the maternal expression of the gene whose cis element is used for driving the reporter gene is suppressed. The zygotic expression of the gene is not suppressed, suggesting that the MASK method can distinguish between maternal and zygotic functions of a gene. Here we investigated the cis and trans factors responsible for MASK results. In the ovaries in which knockdown of a maternal gene occurs, a number of antisense small RNAs are expressed that are complementary to the sequence of the knocked-down genes. We suspect that these antisense small RNAs are the factor responsible for MASK results. The antisense small RNAs have several features that are seen in PIWI-interacting RNAs (piRNAs), suggesting that MASK is likely to use a piRNA-mediated mechanism to knock down maternal mRNAs.