Improvement of reporter activity by IRES-mediated polycistronic reporter system

Many in vitro and in vivo applications for transgenesis require co-expression of heterologous genes. The use of internal ribosome entry sites (IRESs) in dicistronic expression vectors enables the expression of two genes controlled by one promoter in target cells or whole organisms. Here we describe the expansion of IRES exploitation to generate multicistronic vectors capable of expressing multiple reporter genes, especially to improve the fluorescence yield of autofluorescent reporter gene products such as green fluorescent protein (GFP). We found that the increase in fluorescence output of GFP is proportional to the number of IRES-GFP repeats in the multicistronic vector. At least four genes can be expressed from a multicistonic vector by using tandem IRES elements, with no significant alteration of the expression level of the cap-dependent translated gene. Moreover, gene expression under the control of multiple IRES element has no effect on the posttranscriptional regulation through 3′-untranslated regions (3′UTR). Thus, endogenous gene expression and regulation, especially those controlled by weak promoters, can be analyzed with our IRES-dependent polycistronic reporter gene expression system

Etablierung von Interleukin-10 (IL-10)-Reportermäusen zur Identifizierung der IL-10-produzierenden Zellen in vivo

Das Cytokin Interleukin-10 (IL-10) ist eines der wichtigsten Immunsuppressoren, das die Inflammation limitiert und für eine ausgewogene nichtpathologische Immunantwort sorgt. Um die IL-10-produzierenden Zellen in vivo bzw. ex vivo zu identifizieren und IL-10-Genregulation zu analysieren, wurde eine IL-10-IRESEGFP-Reportermaus etabliert. Mit diesem Reportermausmodell konnte EGFP-Expression nur in T-Zellen detektiert werden. Um eine höhere Reportersensitivität zu erreichen, wurden mehrere Reporterkonstrukte generiert und im Zellkultursystem getestet. Mittels IRES-vermittelter polycistronischer Reportervektoren wurde festgestellt, dass die Reporteraktivität proportional zu der Anzahl der IRES-EGFP-Sequenzen ansteigt. Außerdem konnte gezeigt werden, dass die Cap-abhängige Translation und die posttranskriptionale Regulation mittels der 3'-nichttranslatierten Region (3'-UTR) nicht durch multiple IRES-EGFP-Sequenzen beeinträchtigt werden. Um die Reproduzierbarkeit der erzielten Ergebnisse in einem transgenen Mausmodell zu testen, wurde ein polycistronischer Reporter-Targeting-vektor, IL10-3xIRESEGFP, generiert und mittels homologer Rekombination ins Genom von Maus-ES-Zellen integriert. Die IL10-3xIRESEGFP-Reportermaus, die aus den bereits geborenen chimären Mäusen etabliert wird, sollen in weiterführenden Studien umfassend untersucht werden. Um bei dem sehr schwachen IL-10-Promoter eine nachweisbare Reporteraktivität in vivo/ex vivo zu erzielen, wurde in einem zweitem Ansatz das polycistronische Reportersystem mit dem ß-Lactamase-Gen verwendet und eine IL10-2xIRES-Bla-Reportermaus (IL10-Bla-Maus) generiert. Mit dieser Reportermaus konnte die Reporteraktivität wesentlich verbessert werden. Es konnte unter physiologischen Bedingungen die Bla-Reporteraktivität in DCs, Makrophagen, B-Zellen, T-Zellen, NK-Zellen und PMNs, in vitro und ex vivo detektiert werden. Außerdem ermöglicht die IL10-Bla-Reportermaus kinetische Echtzeitexpressionsstudien. Im Unterschied zu IL10-EGFP-Reportermaus, konnte bei im Yersinia-Ifektionsmodell mit der IL10-Bla-Maus Reporteraktivität in verschiedenen Zelltypen ex vivo detektiert werden. Auf der Basis dieser Ergebnisse wurde ein Modell zum zellulären Expressionsprofil von IL-10, bezeichnet als autokrine IL10-abhängige Regulation, vorgeschlagen und die IL-10-Expressionsdaten in diesem Kontext diskutiert

Charakterisierung der asymmetrischen Funktionalität der Nukleotidbindedomänen des ABC-Transporters TAP

Der Transporter associated with antigen processing (TAP) gehört zur Familie der ATP-Binding Cassette (ABC)-Transporter (Higgins 1992). TAP besteht aus zwei Untereinheiten TAP1 und TAP2. Jede Untereinheit besteht aus einer Transmembrandomäne (TMD) und einer Nukleotidbindedomäne (NBD). Sowohl die TMDs als auch die NBDs sind funktional verschieden (Alberts et al. 2001, Daumke et al. 2001, Lapinski et al. 2001). Bei den NBDs beruht die unterschiedliche Funktionalität auf intrinsischen Eigenschaften (Daumke et al. 2001). In dieser Arbeit wurde untersucht, in wieweit die ca. 70 Aminosäuren langen C-termini (V-Regionen) und die Kern-NBDs von TAP (siehe Abbildung I.6.) die asymmetrische Funktionalität der Nukleotidbindedomänen bedingen. Dazu wurden rekombinante Transporter hergestellt, in denen die C-Termini oder die Kern-NBDs zwischen NBD1 und NBD2 wechselseitig ausgetauscht wurden (siehe Abbildung I.6.). Überdies wurde eine weitere TAP2-Mutante, der die C-terminale V-Region fehlt, hergestellt. Die cDNAs für diese chimären Transporter wurden in TAP-negativen T2-Zellen stabil transfiziert. Es wurden insgesamt sechzehn T2-Zelllinien, die stabil ein oder zwei TAP-Ketten exprimieren, etabliert. Bei den Untersuchungen zum Nukleotidbindeverhalten stellte sich heraus, daß der C-Terminus von TAP2 eine für ATP-Bindung ungünstige Konformation in der NBD hervorruft, während der C-Terminus von TAP1 die Bindung von ADP und ATP zulässt. Der C-Terminus von TAP2 (V2) stellt möglicherweise einen limitierenden strukturellen Faktor dar, der die Nukleotidbindung während des Transportzyklus reguliert. Die Bindung von ATP in NBD2 blockiert simultane ATP-Bindung in NBD1, was auf eine strukturelle Wechselwirkung zwischen den beiden NBDs hindeutet. Überdies konnte gezeigt werden, daß die ADP-Bindung eine gegebene intrinsische Eigenschaft der Kern-NBD2 ist. Ein verändertes Verhalten bedingt durch die C-Termini wurde auch bei den Peptidbindeeigenschaften der chimären Transporter festgestellt. Die Untersuchungen zum Peptidbindeverhalten zeigten, dass, unabhängig von der Art der Nukleotidbindung der an die TMDs fusionierten NBDs, ein Einfluss auf die Peptidbindung von den NBDs ausgeht. Die Untersuchungen zur Transportaktivität zeigten, daß nur der Transporter 2/1C2 eine nennenswerte Aktivität aufweist. Dies deutet darauf hin, daß die Kern-NBD2 (C2) die Kern NBD1 (C1) ersetzen kann aber umgekehrt nicht. Das spricht dafür, daß der C-Terminus mit der Kern-NBD in Wechselwirkung tritt, diese Wechselwirkung ist in der hergestellten NBD: C1V2 zerstört. Zur Charakterisierung der entscheidenden Aminosäuren, die für den beobachteten Einfluß des C-Terminus verantwortlichen sein könnten, wurden TAP-cDNAs, in denen die Switch-Regionen zwischen NBD1 und NBD2 ausgetauscht wurden, hergestellt. Diese wurden in TAP-negativen T2-Zellen transfiziert und sollen in weiterführenden Studien charakterisiert werden

Tracing genomic variations in two highly virulent Yersinia enterocolitica strains with unequal ability to compete for host colonization

Background: Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081. Results: Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity < 50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed. Conclusions: Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype

The RNA Chaperone Hfq Is Essential for Virulence and Modulates the Expression of Four Adhesins in Yersinia enterocolitica

In Enterobacteriaceae, the RNA chaperone Hfq mediates the interaction of small RNAs with target mRNAs, thereby modulating transcript stability and translation. This post-transcriptional control helps bacteria adapt quickly to changing environmental conditions. Our previous mutational analysis showed that Hfq is involved in metabolism and stress survival in the enteropathogen Yersinia enterocolitica. In this study we demonstrate that Hfq is essential for virulence in mice and influences production of surface pathogenicity factors, in particular lipopolysaccharide and adhesins mediating interaction with host tissue. Hfq inhibited the production of Ail, the Ail-like protein OmpX and the MyfA pilin post-transcriptionally. In contrast Hfq promoted production of two major autotransporter adhesins YadA and InvA. While protein secretion in vitro was not affected, hfq mutants exhibited decreased protein translocation by the type III secretion system into host cells, consistent with decreased production of YadA and InvA. The influence of Hfq on YadA resulted from a complex interplay of transcriptional, post-transcriptional and likely post-translational effects. Hfq regulated invA by modulating the expression of the transcriptional regulators rovA, phoP and ompR. Therefore, Hfq is a global coordinator of surface virulence determinants in Y. enterocolitica suggesting that it constitutes an attractive target for developing new antimicrobial strategies

Tracing genomic variations in two highly virulent Yersinia enterocolitica strains with unequal ability to compete for host colonization

Background: Yersinia enterocolitica is a gastrointestinal foodborne pathogen found worldwide and which especially affects infants and young children. While different bioserotypes have been associated with varying pathogenicity, research on Y. enterocolitica is mainly conducted on the highly virulent mouse-lethal strains of biotype 1B and serotype O:8. We demonstrate here that two Y. enterocolitica bioserotype 1B/O:8 strains, 8081 and WA-314, display different virulence and fitness properties in a mouse model. In vivo co-infection experiments revealed that strain WA-314 overcomes strain 8081 in the colonization of spleen and liver. To trace the reasons of this incongruity, we present here the first high-quality sequence of the whole genome of strain WA-314 and compare it to the published genome of strain 8081. Results: Regions previously accepted as unique to strain 8081, like the YAPI and YGI-3 genomic islands, are absent from strain WA-314, confirming their strain-specificity. On the other hand, some fitness- and bacterial competition-associated features, such as a putative colicin cluster and a xenobiotic-acyltransferase-encoding gene, are unique to strain WA-314. Additional acquisitions of strain WA-314 are seven prophage-like regions. One of these prophages, the 28-kb P4-like prophage YWA-4, encodes a PilV-like protein that may be used for adhesion to and invasion of the intestinal cells. Furthermore, a putative autotransporter and two type 1 fimbrial proteins of strain WA-314 show a sequence similarity < 50% with the orthologous proteins in strain 8081. The dissimilar sequences of these proteins indicate possible different functions or interaction modes, reflecting the specific adhesion properties of Y. enterocolitica strains 8081 and WA-314 and thus the different efficiency of host colonization. Further important differences were found in two pYV plasmid-encoded virulence factors, YopM and YscP. The impact of these differences on virulence is discussed. Conclusions: Our study emphasizes that the virulence of pathogens can be increased, by acquiring new genes and/or improving the function of essential virulence proteins, resulting in permanently hyper-virulent strains. This work also highlights the importance of addressing genetic and phenotypic variations among closely related bacterial strains, even those belonging to the same bioserotype

A fusion of minicircle DNA and nanoparticle delivery technologies facilitates therapeutic genetic engineering of autologous canine olfactory mucosal cells

Olfactory ensheathing cells (OECs) promote axonal regeneration and improve locomotor function when transplanted into the injured spinal cord. A recent clinical trial demonstrated improved motor function in domestic dogs with spinal injury following autologous OEC transplantation. Their utility in canines offers promise for human translation, as dogs are comparable to humans in terms of clinical management and genetic/environmental variation. Moreover, the autologous, minimally invasive derivation of OECs makes them viable for human spinal injury investigation. Genetic engineering of transplant populations may augment their therapeutic potential, but relies heavily on viral methods which have several drawbacks for clinical translation. We present here the first proof that magnetic particles deployed with applied magnetic fields and advanced DNA minicircle vectors can safely bioengineer OECs to secrete a key neurotrophic factor, with an efficiency approaching that of viral vectors. We suggest that our alternative approach offers high translational potential for the delivery of augmented clinical cell therapies

Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer.

Inhibitors against the p110δ isoform of phosphoinositide-3-OH kinase (PI(3)K) have shown remarkable therapeutic efficacy in some human leukaemias. As p110δ is primarily expressed in leukocytes, drugs against p110δ have not been considered for the treatment of solid tumours. Here we report that p110δ inactivation in mice protects against a broad range of cancers, including non-haematological solid tumours. We demonstrate that p110δ inactivation in regulatory T cells unleashes CD8(+) cytotoxic T cells and induces tumour regression. Thus, p110δ inhibitors can break tumour-induced immune tolerance and should be considered for wider use in oncology

Establishing Biomechanical Mechanisms in Mouse Models: Practical Guidelines for Systematically Evaluating Phenotypic Changes in the Diaphyses of Long Bones

Mice are widely used in studies of skeletal biology, and assessment of their bones by mechanical testing is a critical step when evaluating the functional effects of an experimental perturbation. For example, a gene knockout may target a pathway important in bone formation and result in a “low bone mass” phenotype. But how well does the skeleton bear functional loads; eg, how much do bones deform during loading and how resistant are bones to fracture? By systematic evaluation of bone morphological, densitometric, and mechanical properties, investigators can establish the “biomechanical mechanisms” whereby an experimental perturbation alters whole‐bone mechanical function. The goal of this review is to clarify these biomechanical mechanisms and to make recommendations for systematically evaluating phenotypic changes in mouse bones, with a focus on long‐bone diaphyses and cortical bone. Further, minimum reportable standards for testing conditions and outcome variables are suggested that will improve the comparison of data across studies. Basic biomechanical principles are reviewed, followed by a description of the cross‐sectional morphological properties that best inform the net cellular effects of a given experimental perturbation and are most relevant to biomechanical function. Although morphology is critical, whole‐bone mechanical properties can only be determined accurately by a mechanical test. The functional importance of stiffness, maximum load, postyield displacement, and work‐to‐fracture are reviewed. Because bone and body size are often strongly related, strategies to adjust whole‐bone properties for body mass are detailed. Finally, a comprehensive framework is presented using real data, and several examples from the literature are reviewed to illustrate how to synthesize morphological, tissue‐level, and whole‐bone mechanical properties of mouse long bones. © 2015 American Society for Bone and Mineral ResearchPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/111801/1/jbmr2539.pd

A High-Throughput Platform for Lentiviral Overexpression Screening of the Human ORFeome

In response to the growing need for functional analysis of the human genome, we have developed a platform for high-throughput functional screening of genes overexpressed from lentiviral vectors. Protein-coding human open reading frames (ORFs) from the Mammalian Gene Collection were transferred into lentiviral expression vector using the highly efficient Gateway recombination cloning. Target ORFs were inserted into the vector downstream of a constitutive promoter and upstream of an IRES controlled GFP reporter, so that their transfection, transduction and expression could be monitored by fluorescence. The expression plasmids and viral packaging plasmids were combined and transfected into 293T cells to produce virus, which was then used to transduce the screening cell line. We have optimised the transfection and transduction procedures so that they can be performed using robotic liquid handling systems in arrayed 96-well microplate, one-gene-per-well format, without the need to concentrate the viral supernatant. Since lentiviruses can infect both dividing and non-dividing cells, this system can be used to overexpress human ORFs in a broad spectrum of experimental contexts. We tested the platform in a 1990 gene pilot screen for genes that can increase proliferation of the non-tumorigenic mammary epithelial cell line MCF-10A after removal of growth factors. Transduced cells were labelled with the nucleoside analogue 5-ethynyl-2′-deoxyuridine (EdU) to detect cells progressing through S phase. Hits were identified using high-content imaging and statistical analysis and confirmed with vectors using two different promoters (CMV and EF1α). The screen demonstrates the reliability, versatility and utility of our screening platform, and identifies novel cell cycle/proliferative activities for a number of genes