Target Reporter Gene Research Articles

A Simple Quantitative Assay for Measuring β‐Galactosidase Activity Using X‐Gal in Yeast‐Based Interaction Analyses

Yeast-based interaction assays to determine protein-protein and protein-nucleic acid interactions commonly rely on the reconstitution of chimeric transcription factors that activate the expression of target reporter genes. The enzyme β-galactosidase (β-gal), coded by the LacZ gene of Escherichia coli, is a widely used reporter in yeast systems, and its expression is commonly assessed by evaluating its activity. X-gal (5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside) is an inexpensive and sensitive substrate of β-gal, whose hydrolysis results in an intensely blue colored and easily detectable end product, 5,5'-dibromo-4,4'-dichloro-indigo. The insoluble nature of this end product, however, makes X-gal-based assays unsuitable for direct spectrophotometric absorbance quantification. As such, the use of X-gal is mostly restricted to solid-support approaches, such as colony lift or agar plate assays, which often only provide a qualitative readout. In this article, we describe a quantitative solid-phase X-gal assay to measure protein-protein interaction strength in yeast cells using a simple and low-cost experimental setup. We have optimized multiple aspects of the assay, namely sample preparation, reaction time, and quantification method, for speed and consistency. By integrating the use of a freely available ImageJ-based plugin, we have further standardized the assay for reliability and reproducibility. This improved quantitative X-gal assay can be performed in a standard molecular biology lab without the need for any specialized equipment other than an inexpensive and widely accessible smartphone camera. To exemplify the protocol, we provide detailed step-by-step instructions to perform a quantitative X-gal assay to assess the interaction between two Arabidopsis thaliana proteins, SUPPRESSOR OF PHYA-105 1 (SPA1) and PRODUCTION OF ANTHOCYANIN PIGMENT 2 (PAP2). To demonstrate the sensitivity of our assay in detecting weaker interactions, we also compare the results with a liquid-phase assay that uses ONPG (ortho-nitrophenyl-β-galactopyranoside) as a substrate for β-gal. The quantitative X-gal assay described here can easily be adapted for high-throughout interaction studies and protein domain mapping, even in yeast strains with low levels of LacZ expression. © 2022 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Preparation of competent yeast cells and transformation Alternate Protocol 1: In-house preparation of yeast competent cells for use in lithium acetate (LiAc)-mediated yeast transformation Support Protocol: Long-term storage and revival of frozen yeast strain stocks Basic Protocol 2: Measuring β-galactosidase activity via the quantitative X-gal assay Alternate Protocol 2: Quantification of interaction strength using liquid ONPG assay.

Simultaneous Inhibition of PD-1 and Stimulation of CD40 Signaling Pathways by Anti-PD-L1/CD40L Bispecific Fusion Protein Synergistically Activate Target and Effector Cells

Bispecific antibodies (BsAbs) or fusion proteins (BsAbFPs) present a promising strategy for cancer immunotherapy. Numerous BsAbs targeting coinhibitory and costimulatory pathways have been developed for retargeting T cells and antigen presenting cells (APCs). It is challenging to assess the potency of BsAb that engages two different signaling pathways simultaneously in a single assay format, especially when the two antigen targets are expressed on different cells. To explore the potency of anti-PD-L1/CD40L BsAbFP, a fusion protein that binds to human CD40 and PD-L1, we engineered CHO cells as surrogate APCs that express T cell receptor activator and PD-L1, Jurkat cells with PD-1 and NFAT-luciferase reporter as effector T cells, and Raji cell with NFkB-luciferase that endogenously expresses CD40 as accessory B cells. A novel reporter gene bioassay was developed using these cell lines that allows anti-PD-L1/CD40L BsAbFP to engages both PD-1/PD-L1 and CD40/CD40L signaling pathways in one assay. As both reporters use firefly luciferase, the effects of activating both signaling pathways is observed as an increase in luminescence, either as a higher upper asymptote, a lower EC50, or both. This dual target reporter gene bioassay system reflects potential mechanism of action and demonstrated the ability of anti-PD-L1/CD40L BsAbFP to synergistically induce biological response compared to the combination of anti-PD-L1 monovalent monoclonal antibody and agonist CD40L fusion protein, or either treatment alone. The results also showed a strong correlation between the drug dose and biological response within the tested potency range with good linearity, accuracy, precision, specificity and stability indicating properties, suggesting that this “three-cell-in-one” dual target reporter gene bioassay is suitable for assessing potency, structure-function and critical quality attributes of anti-PD-L1/CD40L BsAbFP. This approach could be used for developing dual target bioassays for other BsAbs and antibodies used for combination therapy.

Mutagenicity in haploid yeast meiosis resulting from repair of DSBs by the sister chromatid.

Mutations in diploid budding yeast occur in meiosis at higher frequencies than in cells grown vegetatively. Such meiotic mutations are thought to result from the repair of double-strand breaks (DSBs) in meiosis, during the process of recombination. Here, we report studies of mutagenicity in haploid strains that may undergo meiosis due to the expression of both mating-type alleles, MATa and MATα. We measure the rate of mutagenicity in the reporter gene CAN1, and find it to be fivefold higher than in mitotic cells, as determined by fluctuation analysis. This enhanced meiotic mutagenicity is shown to depend on the presence of SPO11, the gene responsible for meiotic DSBs. Mutations in haploid meiosis must result from repair of the DSBs through interaction with the sister chromatid, rather than with non-sister chromatids as in diploids. Thus, mutations in diploid meiosis that are not ostensibly associated with recombination events can be explained by sister-chromatid repair. The spectrum of meiotic mutations revealed by Sanger sequencing is similar in haploid and in diploid meiosis. Compared to mitotic mutations in CAN1, long Indels are more frequent among meiotic mutations. Both, meiotic and mitotic mutations are more common at G/C sites than at A/T, in spite of an opposite bias in the target reporter gene. We conclude that sister-chromatid repair of DSBs is a major source of mutagenicity in meiosis.

An RNA Origami Octahedron with Intrinsic siRNAs for Potent Gene Knockdown.

The fields of DNA and RNA nanotechnology have established nucleic acids as valuable building blocks for functional nanodevices with applications in nanomedicine. Here, a simple method for designing and assembling a 3D scaffolded RNA origami wireframe structure with intrinsic functioning small interfering RNAs (siRNAs) embedded is introduced. Uniquely, the method uses an mRNA fragment as scaffold strand, which is folded by sequence-complementarity of nine shorter synthetic strands. High-yield production of the intended 3D structure is verified by transmission electron microscopy (TEM). Production of functional siRNAs is facilitated by incorporating recognition sites for Dicer at selected locations in the structure, and efficient silencing of a target reporter gene is demonstrated.

Design and Experimental Validation of Small Activating RNAs Targeting an Exogenous Promoter in Human Cells.

It is increasingly practical to co-opt many native cellular components into use as elements of synthetic biological systems. We present the design and experimental investigation of the first exogenous genetic construct to be successfully targeted by RNA activation, a phenomenon whereby small double-stranded RNAs increase gene expression from sequence-similar promoters by a mechanism thought to be related to that of RNA interference. Our selection of activating RNA candidates was informed by a custom-written computer program designed to choose target sites in the promoter of interest according to a set of empirical optimality criteria drawn from prior research. Activating RNA candidates were assessed for activity against two exogenously derived target promoters, with successful candidates being subjected to further rounds of validation as a precaution against potential off-target effects. A genetic platform was assembled that allowed activating RNA candidates to be simultaneously screened both for positive activity on the target reporter gene and for possible nonspecific effects on cell metabolism. Several candidate sequences were tested to appraise the utility of this platform, with the most successful achieving a moderate activation level with minimal off-target effects.

Expression Analysis of Hairpin RNA Carrying Sugarcane mosaic virus (SCMV) Derived Sequences and Transgenic Resistance Development in a Model Rice Plant.

Developing transgenic resistance in monocotyledonous crops against pathogens remains a challenging area of research. Sugarcane mosaic virus (SCMV) is a serious pathogen of many monocotyledonous crops including sugarcane. The objective of present study was to analyze transgenic expression of hairpin RNA (hpRNA), targeting simultaneously CP (Coat Protein) and Hc-Pro (helper component-proteinase) genes of SCMV, in a model rice plant. Conserved nucleotide sequences, exclusive for DAG (Aspartic acid-Alanine-Glycine) and KITC (Lycine-Isoleucine-Threonine-Cysteine) motifs, derived from SCMV CP and Hc-Pro genes, respectively, were fused together and assembled into the hpRNA cassette under maize ubiquitin promoter to form Ubi-hpCP:Hc-Pro construct. The same CP:Hc-Pro sequence was fused with the β-glucuronidase gene (GUS) at the 3′ end under CaMV 35S promoter to develop 35S-GUS:CP:Hc-Pro served as a target reporter gene construct. When delivered into rice callus tissues by particle bombardment, the Ubi-hpCP:Hc-Pro construct induced strong silencing of 35S-GUS:CP:Hc-Pro. Transgenic rice plants, containing Ubi-hpCP:Hc-Pro construct, expressed high level of 21–24 nt small interfering RNAs, which induced specific suppression against GUS:CP:Hc-Pro delivered by particle bombardment and conferred strong resistance to mechanically inoculated SCMV. It is concluded that fusion hpRNA approach is an affordable method for developing resistance against SCMV in model rice plant and it could confer SCMV resistance when transformed into sugarcane.

Efficient CRISPR/Cas9-mediated gene editing in Arabidopsis thaliana and inheritance of modified genes in the T2 and T3 generations.

The newly developed CRISPR/Cas9 system for targeted gene knockout or editing has recently been shown to function in plants in both transient expression systems as well as in primary T1 transgenic plants. However, stable transmission of genes modified by the Cas9/single guide RNA (sgRNA) system to the T2 generation and beyond has not been demonstrated. Here we provide extensive data demonstrating the efficiency of Cas9/sgRNA in causing modification of a chromosomally integrated target reporter gene during early development of transgenic Arabidopsis plants and inheritance of the modified gene in T2 and T3 progeny. Efficient conversion of a nonfunctional, out-of-frame GFP gene to a functional GFP gene was confirmed in T1 plants by the observation of green fluorescent signals in leaf tissues as well as the presence of mutagenized DNA sequences at the sgRNA target site within the GFP gene. All GFP-positive T1 transgenic plants and nearly all GFP-negative plants examined contained mutagenized GFP genes. Analyses of 42 individual T2 generation plants derived from 6 different T1 progenitor plants showed that 50% of T2 plants inherited a single T-DNA insert. The efficiency of the Cas9/sgRNA system and stable inheritance of edited genes point to the promise of this system for facile editing of plant genes.

Differential binding of the related transcription factors Pho4 and Cbf1 can tune the sensitivity of promoters to different levels of an induction signal

Transcription factors that belong to the same family typically have similar, but not identical, binding specificities. As such, they can be expected to compete differentially for binding to different variants of their binding sites. Pho4 is a yeast factor whose nuclear concentration is up-regulated in low phosphate, while the related factor, Cbf1, is constitutively expressed. We constructed 16 GFP-reporter genes containing all palindromic variants of the motif NNCACGTGNN, and determined their activities at a range of phosphate concentrations. Pho4 affinity did not explain expression data well except under fully induced conditions. However, reporter activity was quantitatively well explained under all conditions by a model in which Cbf1 itself has modest activating activity, and Pho4 and Cbf1 compete with one another. Chromatin immunoprecipitation and computational analyses of natural Pho4 target genes, along with the activities of the reporter constructs, indicates that genes differ in their sensitivity to intermediate induction signals in part because of differences in their affinity for Cbf1. The induction sensitivity of both natural Pho4 target genes and reporter genes was well explained only by a model that assumes a role for Cbf1 in remodeling chromatin. Our analyses highlight the importance of taking into account the activities of related transcription factors in explaining system-wide gene expression data.

Proteasome inhibition in cancer is associated with enhanced tumor targeting by the adeno-associated virus/phage

Bacteriophage (phage), which are viruses that infect bacteria only, have shown promise as vehicles for targeted cancer gene therapy, albeit with poor efficiency. Recently, we generated an improved version of phage vectors by incorporating cis genetic elements of adeno-associated virus (AAV). This novel AAV/phage hybrid (AAVP) efficiently delivered systemically administered therapeutic genes to various tumor targets by displaying an integrin tumor-targeting ligand on the phage capsid. However, inherent limitations in bacteriophage mean that these AAVP vectors still need to be improved. One of the limitations of AAVP in mammalian cells may be its susceptibility to proteasomal degradation. The proteasome is upregulated in cancer and it is known that it constitutes a barrier to gene delivery by certain eukaryotic viruses. We report here that inhibition of proteasome improved targeted reporter gene delivery by AAVP in cancer cells in vitro and in tumors in vivo after intravenous vector administration to tumor-bearing mice. We also show enhanced targeted tumor cell killing by AAVP upon proteasome inhibition. The AAVP particles persisted significantly in cancer cells in vitro and in tumors in vivo after systemic administration, and accumulated polyubiquitinated coat proteins. Our results suggest that the proteasome is indeed a barrier to tumor targeting by AAVP and indicate that a combination of proteasome-inhibiting drugs and AAVP should be considered for clinical anticancer therapy.

The effect of RAFT-derived cationic block copolymer structure on gene silencing efficiency

In this work a series of ABA tri-block copolymers was prepared from oligo(ethylene glycol) methyl ether methacrylate (OEGMA475) and N,N-dimethylaminoethyl methacrylate (DMAEMA) to investigate the effect of polymer composition on cell viability, siRNA uptake, serum stability and gene silencing. Reversible Addition–Fragmentation Chain Transfer (RAFT) polymerization was used as the method of polymer synthesis as this technique allows the preparation of well-defined block copolymers with low polydispersity. Eight block copolymers were prepared by systematically varying the central cationic block (DMAEMA) length from 38 to 192 monomer units and the outer hydrophilic block (OEGMA475) from 7 to 69 units. The polymers were characterized using size exclusion chromatography and 1H NMR. Chinese Hamster Ovary-GFP and Human Embryonic Kidney 293 cells were used to assay cell viability while the efficiency of block copolymers to complex with siRNA was evaluated by agarose gel electrophoresis. The ability of the polymer–siRNA complexes to enter into cells and to silence the targeted reporter gene enhanced green fluorescent protein (EGFP) was measured by using a CHO-GFP silencing assay. The length of the central cationic block appears to be the key structural parameter that has a significant effect on cell viability and gene silencing efficiency with block lengths of 110–120 monomer units being the optimum. The ABA block copolymer architecture is also critical with the outer hydrophilic blocks contributing to serum stability and overall efficiency of the polymer as a delivery system.

Engineering CHO cell growth by stable manipulation of miRNA expression

Background MiRNAs are small non-coding RNAs involved in many biological functions such as cell proliferation and apoptosis (1), cell cycle (2), homeostasis (3) and cell metabolism (4). They are highly conserved between species. They are capable of regulating hundreds of genes in a post-transcriptional manner by translation repression and/or mRNA degradation (5). These characteristics make miRNAs attractive tools for CHO cell engineering as multiple genes may be targeted simultaneously and possibly entire biological pathways can be manipulated. After temperature-shifting CHO cell culture from 37°C to 31°C, several miRNAs were found differentially regulated of which miR-7 was found to be down-regulated. Our laboratory has previously demonstrated that transient overexpression of miR-7 using mimics of mature endogenous miR-7, leads to a significant decrease in cell growth (6). On the other hand, transient inhibition using inhibitors of mature endogenous miR-7 provoked increased cell growth, though to a lesser extent. As this antisense technology is transient, another strategy was chosen to study the impact of stable miR-7 inhibition on CHO phenotypes. “Sponge” technology is an effective tool to stably sequester endogenous miRNAs (7) by introducing a decoy target reporter gene. This approach can be used to understand post-transcriptional regulation in CHO cells and to identify cellular pathways related to cell growth, cell viability and cell productivity key phenotypes for cell bioprocess improvement.

Platelet Microrna-mRNA Co-Expression Profiles Correlate with Platelet Reactivity

AbstractAbstract 2016 Background:There is extreme inter-individual variation in platelet reactivity, which likely impacts the variation in both risk and clinical outcome of ischemic vascular disease since platelet hyperreactivity has prospectively been shown to be a risk for recurrent coronary syndromes. Although heritability strongly influences the inter-individual variation in platelet reactivity, there is a lack of understanding of the molecular and genetic mechanisms responsible for this variability. To understand some of these mechanisms, we have previously performed mRNA microarray analysis on platelets of subjects with differing levels of platelet reactivity. We showed a differentially expressed (DE) transcript (VAMP8) was associated with platelet reactivity. Intriguingly, we identified a possible role for microRNA (miRNA)-96 in the regulation of VAMP8 mRNA and protein expression. MiRNAs regulate numerous aspects of normal cell physiology and cause disease by altering protein expression, and recent data demonstrate a role for miRNAs in both normal and diseased human megakaryocytopoiesis. Although others and we have observed miRNAs in platelets, their biology is largely unexplored. Aims:To test whether platelet miRNA levels were associated with platelet reactivity in 19 healthy subjects. Because we had previously obtained platelet mRNA profile data on these 19 subjects, we also had a unique opportunity to test for relationships between differentially expressed miRNAs and target DE mRNAs. Methods:MiRNA microarray analysis was performed on leukocyte depleted platelets from 19 healthy subjects with marked variability in platelet responsiveness. Bioinformatics approaches were used analyze the miRNA data in platelets. Subsequently transfection experiments in cell lines to assess miRNA knockdown of target gene products and reporter gene assays were used for functional assessment of miRNA binding to 3'UTRs of the target genes. Results:We found that human platelets express 284 miRNAs, some at very high levels. Unsupervised hierarchical clustering of miRNA profiles resulted in two groups of subjects that appeared to cluster by platelet aggregation phenotypes. Seventy-four miRNAs were differentially expressed between subjects grouped according to platelet aggregation to epinephrine, a subset of which predicted the platelet reactivity response. We profiled miRNAs from HEL and Meg-01 cells and found a strong correlation between normal human platelets and both HEL cells and Meg-01 cells. Using whole-genome mRNA expression data on these same 19 subjects, we computationally generated a high-priority list of miRNA-mRNA pairs where the differentially expressed platelet miRNAs had binding sites in 3'UTRs of differentially expressed mRNAs, and the levels were negatively correlated. From this list, three miRNA-mRNA pairs (miR-200b:PRKAR2B, miR-495:KLHL5 and miR-107:CLOCK) were selected, and all three miRNAs knocked down the protein expression of the target mRNA. Co-transfection experiments using reporter gene constructs engineered to contain the candidate mRNA 3'UTR and corresponding miRNA demonstrated that the miRNA of interest directly targeted the 3'UTR of the candidate mRNA. Conclusions:Results from this study demonstrated (1) platelet miRNAs are able to repress expression of platelet proteins; (2) platelet miRNA profiles are associated with and may predict platelet reactivity and (3) bioinformatic approaches can successfully identify functional miRNAs in platelets. Our findings suggest that selected platelet miRNAs may have potential as biomarkers for vascular thrombosis. It will be important to consider the repertoire and levels of miRNAs when attempting to elucidate the molecular mechanisms responsible for inter-individual variation in platelet reactivity. Disclosures:No relevant conflicts of interest to declare.

Lin28 recruits the TUTase Zcchc11 to inhibit let-7 maturation in mouse embryonic stem cells

Lin28 and Lin28B, two developmentally regulated RNA-binding proteins and proto-oncogenes, selectively inhibit the maturation of let-7 family miRNAs in embryonic stem (ES) cells and certain cancers. Moreover, let-7 precursors (pre-let-7) were previously found to be terminally uridylated in a Lin28-dependent fashion. Here, we identify Zcchc11 (zinc finger, CCHC domain containing 11) as the 3′ terminal uridylyl transferase (TUTase) responsible for Lin28-mediated pre-let-7 uridylation and blockade of let-7 processing in mouse ES cells. We demonstrate that Zcchc11 activity is UTP-dependent, selective for let-7, and recruited by Lin28. Furthermore, knockdown of either Zcchc11 or Lin28, or overexpression of a catalytically inactive TUTase, relieves the selective inhibition of let-7 processing and leads to the accumulation of mature let-7 miRNAs and repression of let-7 target reporter genes. Our results establish a novel role for Zcchc11-catalyzed pre-let-7 uridylation in the control of miRNA biogenesis.

Selection of cell death‐deficient p53 mutants in Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae is a useful system for the detection and transcriptional evaluation of mutant p53 in cancer. In previous work we showed that the overexpression of wild-type p53 induces yeast cell death on minimal medium; however, the R248W p53 mutant was completely inactive, and we suggested that ROS production is a key event in p53-induced yeast cell death. In this study we explored the effect of other p53 mutants, such as the hot-spot mutant R282W and the double mutant N268S::I332V. Unexpectedly, both mutants behaved inversely to R248W, as they completely inhibited yeast growth on minimal medium and induced ROS production. This phenotype 'yeast cell death on minimal medium' allowed for the subsequent screening of intragenic p53-inactivating mutations. In all cases, the 'revertant yeast clones' display a complete p53 inactivation through either gross deletion or nonsense mutations. More interestingly, missense mutations were also found: the deletion of I255 or substitution of R337G completely inactivated the p53 mutant R282W in the yeast context. Taken together, these results suggest that p53 tumour-derived mutants could be classified according to their ability to induce yeast cell death and not uniquely by their transcriptional activity on a selected target reporter gene.

Functional resolution of duplicatedhoxb5genes in teleosts

The duplication-degeneration-complementation (DDC) model predicts that subfunctionalization of duplicated genes is a common mechanism for their preservation. The additional Hox complexes of teleost fish constitute a good system in which to test this hypothesis. Zebrafish have two hoxb complexes, with two hoxb5 genes, hoxb5a and hoxb5b, the expression patterns of which suggest subfunctionalization of an ancestral hoxb5 gene. We characterized conserved non-coding elements (CNEs) near the zebrafish hoxb5 genes. One CNE, J3, is only retained in the hoxb5a locus, whereas the others, J1 and J2, are present in both hoxb5 loci. When tested individually, the enhancer activity of individual CNEs, including J3, extensively overlapped and did not support a role in subfunctionalization. By contrast, reporter transgene constructs encompassing multiple CNEs were able to target reporter gene expression to unique domains of hoxb5a and hoxb5b expression. The deletion of J3 from the hoxb5a locus resulted in expression that approached that of hoxb5b, whereas its insertion in the hoxb5b locus increased reporter expression and rendered it more similar to that of hoxb5a. Our results highlight the importance of interactions between CNEs in the execution of complementary subfunctions of duplicated genes.

Variation of gene silencing involving endogenous microRNA in mammalian cells

MicroRNAs (miRNAs) are small noncoding RNA and play a role in gene expression regulation by inhibiting translation of their target messenger RNAs (mRNAs). In this study, we investigated the effects of endogenous let-7 miRNA on the expression of target genes in various mammalian cells by means of two types of reporter plasmids possessing target sequences for let-7: one carries perfectly matched target sequence for let-7 in the 3'-untranslated region of the luciferase reporter gene to monitor RNA interference (RNAi) activity and the other has three bulged binding sites for let-7 to monitor translation-inhibition activity. The results indicate that different cells have different levels of gene silencing against the target reporter genes. The data presented here suggest that not only microRNA level but also target transcript level likely participate in the generation of a variety of gene silencing.

Construction of a recombinant Pim3-expressing plasmid and expression and activity thereof: experiment with rats

To construct a plasmid expressing green fluorescent protein (GFP) and gene of Pim-3, a member of the serine/threonine kinase family, and to investigate the in vivo expression of the construct and its effect on cell apoptosis. Pim-3 gene was cloned from myocardium tissues of Wistar rat by RT-PCR and subcloned into GFP-expressing plasmid vector pEGFP-N2 by restriction enzyme. The recombinant plasmid pEGFP-N2/Pim-3 was constructed by T4-ligase and then identified through enzyme digestion and gene sequencing. Thirty-two Wistar rats were randomly divided into 4 equal groups: Group A, as control group; Group B, injected intravenously with Ringer's solution; Group C, injected with blank vector, and Group D, injected with the recombinant plasmid pEGFP-N2/Pim-3. One day later, endotoxin/D-galactoamine (D-GalN) was intraperitoneally injected. 24 hours later the rats were killed. Fluorescence microscopy was used to observe the expression of the reporter gene GFP in the liver tissues. RT-PCR was used to detect the Pim-3 mRNA expression. The hepatic apoptosis was detected by TUNEL assay. The activity of caspase-3 was detected. A 998 bp target cDNA fragment with restriction enzyme sites was amplified and inserted into the multiple clone site of pEGFP-N2 successfully. High expression levels of the target gene Pim-3 and reporter gene GFP were achieved in the rat liver after transfer of the recombinant plasmid. The relative Pim-3 expression level of Group D was 0.49 +/- 0.15, significantly higher than those of Groups A, B, and C (0.06 +/- 0.02, 0, and 0 respectively, all P < 0.01). The apoptotic index of Group D was (4.9 +/- 1.2)%, significantly lower than those of Groups B and C [(72.5 +/- 6.1)% and (69.8 +/- 5.7)% respectively, both P < 0.01]; however, not significantly different from that of Group A [(3.1 +/- 0.7)%]. The activity of caspase-3 of Group D was (76 +/- 27) pmol.min(-1).mg(-1), significantly lower than those of Groups B and C [(147 +/- 55) and (142 +/- 50) pmol.min(-1).mg(-1), respectively, both P < 0.01]; however, not significantly different from that of Group A (60 +/- 15). The recombinant plasmid pEGFP-N2/Pim-3 can achieve high expression in living cells and have an inhibitory effect on hepatic apoptosis.

The EWS–Oct-4 fusion gene encodes a transforming gene

The t(6;22)(p21;q12) translocation associated with human bone and soft-tissue tumours results in a chimaeric molecule fusing the NTD (N-terminal domain) of the EWS (Ewing's sarcoma) gene to the CTD (C-terminal domain) of the Oct-4 (octamer-4) embryonic gene. Since the N-terminal domains of EWS and Oct-4 are structurally different, in the present study we have assessed the functional consequences of the EWS-Oct-4 fusion. We find that this chimaeric gene encodes a nuclear protein which binds DNA with the same sequence specificity as the parental Oct-4 protein. Comparison of the transactivation properties of EWS-Oct-4 and Oct-4 indicates that the former has higher transactivation activity for a known target reporter gene containing Oct-4 binding. Deletion analysis of the functional domains of EWS-Oct-4 indicates that the EWS (NTD), the POU domain and the CTD of EWS-Oct-4 are necessary for full transactivation potential. EWS-Oct-4 induced the expression of fgf-4 (fibroblast growth factor 4) and nanog, which are potent mitogens as well as Oct-4 downstream target genes whose promoters contain potential Oct-4-binding sites. Finally, ectopic expression of EWS-Oct-4 in Oct-4-null ZHBTc4 ES (embryonic stem) cells resulted in increased tumorigenic growth potential in nude mice. These results suggest that the oncogenic effect of the t(6;22) translocation is due to the EWS-Oct-4 chimaeric protein and that fusion of the EWS NTD to the Oct-4 DNA-binding domain produces a transforming chimaeric product.

Transcriptional Regulation of Pituitary POMC Is Conserved at the Vertebrate Extremes Despite Great Promoter Sequence Divergence

The stress response involves complex physiological mechanisms that maximize behavioral efficacy during attack or defense and is highly conserved in all vertebrates. Key mediators of the stress response are pituitary hormones encoded by the proopiomelanocortin gene (POMC). Despite conservation of physiological function and expression pattern of POMC in all vertebrates, phylogenetic footprinting analyses at the POMC locus across vertebrates failed to detect conserved noncoding sequences with potential regulatory function. To investigate whether ortholog POMC promoters from extremely distant vertebrates are functionally conserved, we used 5'-flanking sequences of the teleost fish Tetraodon nigroviridis POMCalpha gene to produce transgenic mice. Tetraodon POMCalpha promoter targeted reporter gene expression exclusively to mouse pituitary cells that normally express Pomc. Importantly, transgenic expression in mouse corticotrophs was increased after adrenalectomy. To understand how conservation of precise gene expression mechanisms coexists with great sequence divergence, we investigated whether very short elements are still conserved in all vertebrate POMC promoters. Multiple local sequence alignments that consider phylogenetic relationships of ortholog regions identified a unique 10-bp motif GTGCTAA(T/G)CC that is usually present in two copies in POMC 5'-flanking sequences of all vertebrates. Underlined nucleotides represent totally conserved sequences. Deletion of these paired motifs from Tetraodon POMCalpha promoter markedly reduced its transcriptional activity in a mouse corticotropic cell line and in pituitary POMC cells of transgenic mice. In mammals, the conserved motifs correspond to reported binding sites for pituitary-specific nuclear proteins that participate in POMC transcriptional regulation. Together, these results demonstrate that mechanisms that participate in pituitary-specific and hormonally regulated expression of POMC have been preserved since mammals and teleosts diverged from a common ancestor 450 million years ago despite great promoter sequence divergence.

In vivo gene silencing in solid tumors by targeted electrically mediated siRNA delivery

RNA interference (RNAi)-mediated gene silencing approaches appear very promising for therapies based on the targeted inhibition of disease-relevant genes. The major hurdle to the therapeutic development of RNAi strategies remains, however, the efficient delivery of the RNAi-inducing molecules, the short interfering RNAs (siRNAs) and short hairpin RNAs (shRNAs), to the target tissue. With respect to cancer treatment the development of efficient delivery methods into solid tumors appears as a critical issue. However, very few studies have addressed this problem. In this study we have investigated the contribution of electrically mediated delivery of siRNA into murine tumors stably expressing an enhanced green fluorescent protein (EGFP) target reporter gene. The silencing of EGFP gene expression was quantified over time by fluorescence imaging in the living animal. Our study indicates that electric field can be used as an efficient method for siRNA delivery and associated gene silencing into cells of solid tumors in vivo.