Enhanced gene expression from retroviral vectors

<p>Abstract</p> <p>Background</p> <p>Retroviruses are widely used to transfer genes to mammalian cells efficiently and stably. However, genetic elements required for high-level gene expression are incompatible with standard systems. The retroviral RNA genome is produced by cellular transcription and post-transcriptional processing within packaging cells: Introns present in the retroviral genomic transcript are removed by splicing, while polyadenylation signals lead to the production of ineffective truncated genomes. Furthermore strong enhancer/promoters within the retroviral payload lead to detrimental competition with the retroviral enhancer/promoter.</p> <p>Results</p> <p>By exploiting a new method of producing the retroviral genome <it>in vitro </it>it is possible to produce infectious retroviral particles carrying a high-level expression cassette that completely prohibits production of infectious retroviral particles by conventional methods.</p> <p>We produced an expression cassette comprising a strong enhancer/promoter, an optimised intron, the GFP open reading frame and a strong polyadenylation signal. This cassette was cloned into both a conventional MMLV retroviral vector and a vector designed to allow <it>in vitro </it>transcription of the retroviral genome by T7 RNA polymerase.</p> <p>When the conventional retroviral vector was transfected into packaging cells, the expression cassette drove strong GFP expression, but no infectious retrovirus was produced. Introduction of the <it>in vitro </it>produced uncapped retroviral genomic transcript into the packaging cells did not lead to any detectable GFP expression. However, infectious retrovirus was easily recovered, and when used to infect target primary human cells led to very high GFP expression – up to 3.5 times greater than conventional retroviral LTR-driven expression.</p> <p>Conclusion</p> <p>Retroviral vectors carrying an optimized high-level expression cassette do not produce infectious virions when introduced into packaging cells by transfection of DNA. Infectious retrovirus carrying the same cassette is readily produced when packaging cells are transfected with <it>in vitro </it>transcribed retroviral genomic RNA. The applications of this technique are not limited to producing the higher levels of transgene expression demonstrated here. For example, novel reporters with alternatively spliced exon-intron configurations could readily be transduced into virtually any cell. Furthermore, because the <it>in vitro </it>transcripts are not translated within the packaging cells, retroviruses carrying genes lethal to the packaging cells can also be produced.</p

Identification and analysis of serpin-family genes by homology and synteny across the 12 sequenced Drosophilid genomes.

BACKGROUND: The Drosophila melanogaster genome contains 29 serpin genes, 12 as single transcripts and 17 within 6 gene clusters. Many of these serpins have a conserved "hinge" motif characteristic of active proteinase inhibitors. However, a substantial proportion (42%) lacks this motif and represents non-inhibitory serpin-fold proteins of unknown function. Currently, it is not known whether orthologous, inhibitory serpin genes retain the same target proteinase specificity within the Drosophilid lineage, nor whether they give rise to non-inhibitory serpin-fold proteins or other, more diverged, proteins. RESULTS: We collated 188 orthologues to the D. melanogaster serpins from the other 11 Drosophilid genomes and used synteny to find further family members, raising the total to 226, or 71% of the number of orthologues expected assuming complete conservation across all 12 Drosophilid species. In general the sequence constraints on the serpin-fold itself are loose. The critical Reactive Centre Loop (RCL) sequence, including the target proteinase cleavage site, is strongly conserved in inhibitory serpins, although there are 3 exceptional sets of orthologues in which the evolutionary constraints are looser. Conversely, the RCL of non-inhibitory serpin orthologues is less conserved, with 3 exceptions that presumably bind to conserved partner molecules. We derive a consensus hinge motif, for Drosophilid inhibitory serpins, which differs somewhat from that of the vertebrate consensus. Three gene clusters appear to have originated in the melanogaster subgroup, Spn28D, Spn77B and Spn88E, each containing one inhibitory serpin orthologue that is present in all Drosophilids. In addition, the Spn100A transcript appears to represent a novel serpin-derived fold. CONCLUSION: In general, inhibitory serpins rarely change their range of proteinase targets, except by a duplication/divergence mechanism. Non-inhibitory serpins appear to derive from inhibitory serpins, but not the reverse. The conservation of different family members varied widely across the 12 sequenced Drosophilid genomes. An approach considering synteny as well as homology was important to find the largest set of orthologues.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

The impact of quantitative optimization of hybridization conditions on gene expression analysis.

BACKGROUND: With the growing availability of entire genome sequences, an increasing number of scientists can exploit oligonucleotide microarrays for genome-scale expression studies. While probe-design is a major research area, relatively little work has been reported on the optimization of microarray protocols. RESULTS: As shown in this study, suboptimal conditions can have considerable impact on biologically relevant observations. For example, deviation from the optimal temperature by one degree Celsius lead to a loss of up to 44% of differentially expressed genes identified. While genes from thousands of Gene Ontology categories were affected, transcription factors and other low-copy-number regulators were disproportionately lost. Calibrated protocols are thus required in order to take full advantage of the large dynamic range of microarrays.For an objective optimization of protocols we introduce an approach that maximizes the amount of information obtained per experiment. A comparison of two typical samples is sufficient for this calibration. We can ensure, however, that optimization results are independent of the samples and the specific measures used for calibration. Both simulations and spike-in experiments confirmed an unbiased determination of generally optimal experimental conditions. CONCLUSIONS: Well calibrated hybridization conditions are thus easily achieved and necessary for the efficient detection of differential expression. They are essential for the sensitive pro filing of low-copy-number molecules. This is particularly critical for studies of transcription factor expression, or the inference and study of regulatory networks.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Bone marrow cell trafficking following intravenous administration

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/75122/1/j.1365-2141.1999.01779.x.pd

Identification of multiple transcription factor genes potentially involved in the development of electrosensory versus mechanosensory lateral line organs

In electroreceptive jawed vertebrates, embryonic lateral line placodes give rise to electrosensory ampullary organs as well as mechanosensory neuromasts. Previous reports of shared gene expression suggest that conserved mechanisms underlie electroreceptor and mechanosensory hair cell development and that electroreceptors evolved as a transcriptionally related “sister cell type” to hair cells. We previously identified only one transcription factor gene, Neurod4, as ampullary organ-restricted in the developing lateral line system of a chondrostean ray-finned fish, the Mississippi paddlefish (Polyodon spathula). The other 16 transcription factor genes we previously validated in paddlefish were expressed in both ampullary organs and neuromasts. Here, we used our published lateral line organ-enriched gene-set (arising from differential bulk RNA-seq in late-larval paddlefish), together with a candidate gene approach, to identify 25 transcription factor genes expressed in the developing lateral line system of a more experimentally tractable chondrostean, the sterlet (Acipenser ruthenus, a small sturgeon), and/or that of paddlefish. Thirteen are expressed in both ampullary organs and neuromasts, consistent with conservation of molecular mechanisms. Seven are electrosensory-restricted on the head (Irx5, Irx3, Insm1, Sp5, Satb2, Mafa and Rorc), and five are the first-reported mechanosensory-restricted transcription factor genes (Foxg1, Sox8, Isl1, Hmx2 and Rorb). However, as previously reported, Sox8 is expressed in ampullary organs as well as neuromasts in a catshark (Scyliorhinus canicula), suggesting the existence of lineage-specific differences between cartilaginous and ray-finned fishes. Overall, our results support the hypothesis that ampullary organs and neuromasts develop via largely conserved transcriptional mechanisms, and identify multiple transcription factors potentially involved in the formation of electrosensory versus mechanosensory lateral line organs

AXL targeting restores PD-1 blockade sensitivity of STK11/LKB1 mutant NSCLC through expansion of TCF1+ CD8 T cells

Mutations in STK11/LKB1 in non-small cell lung cancer (NSCLC) are associated with poor patient responses to immune checkpoint blockade (ICB), and introduction of a Stk11/Lkb1 (L) mutation into murine lung adenocarcinomas driven by mutant Kras and Trp53 loss (KP) resulted in an ICB refractory syngeneic KPL tumor. Mechanistically this occurred because KPL mutant NSCLCs lacked TCF1-expressing CD8 T cells, a phenotype recapitulated in human STK11/LKB1 mutant NSCLCs. Systemic inhibition of Axl results in increased type I interferon secretion from dendritic cells that expanded tumor-associated TCF1+PD-1+CD8 T cells, restoring therapeutic response to PD-1 ICB in KPL tumors. This was observed in syngeneic immunocompetent mouse models and in humanized mice bearing STK11/LKB1 mutant NSCLC human tumor xenografts. NSCLC-affected individuals with identified STK11/LKB1 mutations receiving bemcentinib and pembrolizumab demonstrated objective clinical response to combination therapy. We conclude that AXL is a critical targetable driver of immune suppression in STK11/LKB1 mutant NSCLC.publishedVersio

Mural Cell Associated VEGF Is Required for Organotypic Vessel Formation

Background: Blood vessels comprise endothelial cells, mural cells (pericytes/vascular smooth muscle cells) and basement membrane. During angiogenesis, mural cells are recruited to sprouting endothelial cells and define a stabilizing context, comprising cell-cell contacts, secreted growth factors and extracellular matrix components, that drives vessel maturation and resistance to anti-angiogenic therapeutics. Methods and Findings: To better understand the basis for mural cell regulation of angiogenesis, we conducted high content imaging analysis on a microtiter plate format in vitro organotypic blood vessel system comprising primary human endothelial cells co-cultured with primary human mural cells. We show that endothelial cells co-cultured with mural cells undergo an extensive series of phenotypic changes reflective of several facets of blood vessel formation and maturation: Loss of cell proliferation, pathfinding-like cell migration, branching morphogenesis, basement membrane extracellular matrix protein deposition, lumen formation, anastamosis and development of a stabilized capillary-like network. This phenotypic sequence required endothelial-mural cell-cell contact, mural cell-derived VEGF and endothelial VEGFR2 signaling. Inhibiting formation of adherens junctions or basement membrane structures abrogated network formation. Notably, inhibition of mural cell VEGF expression could not be rescued by exogenous VEGF. Conclusions: These results suggest a unique role for mural cell-associated VEGF in driving vessel formation and maturation

The 3rd DBCLS BioHackathon: improving life science data integration with Semantic Web technologies.

BACKGROUND: BioHackathon 2010 was the third in a series of meetings hosted by the Database Center for Life Sciences (DBCLS) in Tokyo, Japan. The overall goal of the BioHackathon series is to improve the quality and accessibility of life science research data on the Web by bringing together representatives from public databases, analytical tool providers, and cyber-infrastructure researchers to jointly tackle important challenges in the area of in silico biological research. RESULTS: The theme of BioHackathon 2010 was the 'Semantic Web', and all attendees gathered with the shared goal of producing Semantic Web data from their respective resources, and/or consuming or interacting those data using their tools and interfaces. We discussed on topics including guidelines for designing semantic data and interoperability of resources. We consequently developed tools and clients for analysis and visualization. CONCLUSION: We provide a meeting report from BioHackathon 2010, in which we describe the discussions, decisions, and breakthroughs made as we moved towards compliance with Semantic Web technologies - from source provider, through middleware, to the end-consumer.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Identification of functional elements and regulatory circuits by Drosophila modENCODE

To gain insight into how genomic information is translated into cellular and developmental programs, the Drosophila model organism Encyclopedia of DNA Elements (modENCODE) project is comprehensively mapping transcripts, histone modifications, chromosomal proteins, transcription factors, replication proteins and intermediates, and nucleosome properties across a developmental time course and in multiple cell lines. We have generated more than 700 data sets and discovered protein-coding, noncoding, RNA regulatory, replication, and chromatin elements, more than tripling the annotated portion of the Drosophila genome. Correlated activity patterns of these elements reveal a functional regulatory network, which predicts putative new functions for genes, reveals stage- and tissue-specific regulators, and enables gene-expression prediction. Our results provide a foundation for directed experimental and computational studies in Drosophila and related species and also a model for systematic data integration toward comprehensive genomic and functional annotation

Finishing the euchromatic sequence of the human genome

The sequence of the human genome encodes the genetic instructions for human physiology, as well as rich information about human evolution. In 2001, the International Human Genome Sequencing Consortium reported a draft sequence of the euchromatic portion of the human genome. Since then, the international collaboration has worked to convert this draft into a genome sequence with high accuracy and nearly complete coverage. Here, we report the result of this finishing process. The current genome sequence (Build 35) contains 2.85 billion nucleotides interrupted by only 341 gaps. It covers ∼99% of the euchromatic genome and is accurate to an error rate of ∼1 event per 100,000 bases. Many of the remaining euchromatic gaps are associated with segmental duplications and will require focused work with new methods. The near-complete sequence, the first for a vertebrate, greatly improves the precision of biological analyses of the human genome including studies of gene number, birth and death. Notably, the human enome seems to encode only 20,000-25,000 protein-coding genes. The genome sequence reported here should serve as a firm foundation for biomedical research in the decades ahead