Approaches to Minimize Variation of Transgene Expression in Plants | SpringerLink
Genetic transformation of plants has become a widely used technology that serves multiple purposes in plant biology research. However, considerable variation of transgene expression is often observed within populations of transgenic plants transformed with the same transgene construct. This inter-transformant variation of transgene expression hampers proper evaluation of transgenes and might be most undesirable when high-throughput transgene screening is intended. The general plant transformation strategy today is to generate a sufficiently high number of transgenic plants to find some transformants with the desired level of expression. To reduce cost, labor and interpretational flaws, multiple efforts are being directed toward achieving stable expression of transgenes with an expected level of expression. Various factors are thought to contribute to transgene expression variation including the transgene copy number, RNA silencing, transgene insertion site and the employment ...https://link.springer.com/article/10.1007%2Fs11032-005-4929-9
Stacking Multiple Transgenes at a Selected Genomic Site via Repeated Recombinase-Mediated DNA Cassette Exchanges | Plant...
Figure 5. Analysis of the second round RMCE event. PCR assays specific to the genomic borders and internal regions of the second RMCE DNA, QC288A436A438A, were done on RMCE T0 plant B531-1 using various primer combinations (Fig. 2). The hemizygous (RMCE/excision) B531-1 ancestors hemizygous B53, homozygous B5 and B, and wild-type DNA (wt) were included as controls. A, The expected 886-bp 5′ border of both QC288A in B and QC288A436A in B53 (left) and the 561-bp 3′ border of QC288A in B (center) were amplified. The full-length 4,742-bp QC288A of B, 6,331-bp QC288A329A of B5, and 986-bp QC288ME (excision) of B53 and B531 were amplified (right). The expected full-length 9,108-bp QC288A436A of B53 and 21,925-bp QC288A436A438A of B531-1 were too large to be amplified. B, The expected 967-bp 5′ border of both QC288A329A in B5 and QC288A436A438A in B531 (left) and the 1,180-bp 3′ border of QC288A329A in B5 (center) were amplified. The same B5 band was also amplified from B53 that contained ...http://www.plantphysiol.org/content/154/2/622/tab-figures-data
Dissecting enhancer/promoter function for high-level, persistent transgene expression - Radcliffe Department of Medicine
Unscramble Words that Start With transgenes | Scrabble Finder Words that Begin with transgenes
Words that start with transgenes, Unscrambled words that start with transgenes, Words starting with transgenes, Words that begin with transgenes, Words beginning with transgenes, Words with the prefix transgeneshttp://www.allscrabblewords.com/words-that-start-with/transgenes
Read alignments surrounding transgene integration.The s | Open-i
Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) disphttps://openi.nlm.nih.gov/detailedresult.php?img=PMC4559457_pone.0136817.g003&req=4
3. Venault, A., Y. C. Huang, J. W. Lo, Chung-Jung Chou, A. Chinnathambi, A. Higuchi, W. S. Chen, W. Y. Chen and Y. Chang (2017). Tunable PEGylation of Branch-type PEI/DNA Polyplexes with a Compromise of Low Cytotoxicity and High Transgene Expression: in vitro and in vivo Gene Delivery.'JOURNAL OF MATERIALS CHEMISTRY B 5(24): 4732-4744 ...http://che.cycu.edu.tw/index.php?a=member/page&id=70
Biology | Free Full-Text | Targeted Toxin-Based Selectable Drug-Free Enrichment of Mammalian Cells with High Transgene...
Almost all transfection protocols for mammalian cells use a drug resistance gene for the selection of transfected cells. However, it always requires the characterization of each isolated clone regarding transgene expression, which is time-consuming and labor-intensive. In the current study, we developed a novel method to selectively isolate clones with high transgene expression without drug selection. Porcine embryonic fibroblasts were transfected with pCEIEnd, an expression vector that simultaneously expresses enhanced green fluorescent protein (EGFP) and endo-b-galactosidase C(EndoGalC; an enzyme capable of digesting cell surface a-Gal epitope) upon transfection. After transfection, the surviving cells were briefly treated with IB4SAP (a-Gal epitope-specific BS-I-B4 lectin conjugated with a toxin saporin). The treated cells were then allowed to grow in normal medium, during which only cells strongly expressing EndoGalC and EGFP would survive because of the absence of a-Gal epitopes on their cellhttp://mdpi.com/2079-7737/2/1/341/xml
Transgenic trees currently are being produced by Agrobacterium-mediated transformation and biolistics. Since trees are particularly suited for long-term evaluations of the impact of the technology, Prunus subhirtella autumnorosa (PAR) was chosen as model fruit tree species and transformed with a reporter gene (uidA) under the control of the 35S promoter. Using Southern and GUS fluorometric techniques, we compared transgene copy numbers and observed stability of transgene expression levels in 34 different transgenic plants, grown under in vitro, greenhouse and screenhouse conditions, over a period of 9 years. An influence of grafting on gene expression was not observed. No silenced transgenic plant was detected. Overall, these results suggest that transgene expression in perennial species, such as fruit trees, remains stable in time and space, over extended periods and in different organs, confirming the value of PAR as model species to study season-dependent regulation in mature stonefruit ...https://genera.biofortified.org/view/Maghuly2007
Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles...
Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silencedhttp://www.readabstracts.com/Biological-sciences/Transgene-integration-into-the-same-chromosome-location-can-produce-alleles-that-express-at-a-predic.html
Which cell lines are best for transient transgene expression? - Cell Biology - BioForum
Which cell lines are best for transient transgene expression? - posted in Cell Biology: Hello, I was wondering if anyone has advice regarding which cell lines most efficiently express transgenes following transient transfection. I am not asking which cell lines are most efficiently transfected, but rather which cell lines express genes to the highest levels once they are transfected (although I realize that transfection efficiency and transgene expression efficiency may be related). Than...http://www.protocol-online.org/forums/topic/14522-which-cell-lines-are-best-for-transient-transgene-expression/
British Library EThOS: Contribution of CpG islands to ubiquitous gene expression
The effect of CpG islands on transgene expression was first tested in cultured cells. In transient transfection it was demonstrated that CpG islands do not influence the expression of a transgene when not integrated into the genome. Even when integrated into the genome of cultured cells, CpG islands are not able to confer position-independent, copy number-dependent transgene expression, as confirmed by the analysis of individual cell lines. However, the results from bulk analysis of primary clones suggest that CpG islands improve the level of expression in cultured cells, and increase the proportion of highly expressing clones. Transgenic mice were used to study the effect of CpG islands on the level and pattern of transgene expression in vivo. Unexpectedly, from the nine transgenic lines generated, transgene expression was detected in only one line. In the rest of the lines transgene expression was silenced, and in these cases the transgene was densely methylated. In half of the silenced lines ...http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.654021
A safe and effective gene therapeutic strategy for cancer depends greatly on 2 key determinants: tumor specificity and efficient transgene expression. With the goal of developing a cancer-specific vector with robust activity, we selected the hTERT promoter. Telomerase activation is observed in approximately 90% of human cancers, irrespective of tumor type, whereas most normal tissues contain inactivated telomerase (26), and its promoter has been used in many types of cancer, including ovarian and breast (14, 16). Our data showed that the minimal promoter fragment of hTERT is active in both ADPC and CRPC cells but has much lower activity than CMV promoter. To overcome the lack of strong activities in tissue-specific promoters, we used VISA expression system that has been successfully applied to other cancer types (11, 13-16).. In most cases of recurrent or metastatic prostate cancer through ADPC to CRPC progression, the AR gene is amplified and/or overexpressed and binds to androgen (or androgen ...http://mct.aacrjournals.org/content/13/7/1813
BAC TransgeneOmics: a high-throughput method for exploration of protein function in mammals. - Oxford Neuroscience
The interpretation of genome sequences requires reliable and standardized methods to assess protein function at high throughput. Here we describe a fast and reliable pipeline to study protein function in mammalian cells based on protein tagging in bacterial artificial chromosomes (BACs). The large size of the BAC transgenes ensures the presence of most, if not all, regulatory elements and results in expression that closely matches that of the endogenous gene. We show that BAC transgenes can be rapidly and reliably generated using 96-well-format recombineering. After stable transfection of these transgenes into human tissue culture cells or mouse embryonic stem cells, the localization, protein-protein and/or protein-DNA interactions of the tagged protein are studied using generic, tag-based assays. The same high-throughput approach will be generally applicable to other model systems.https://www.neuroscience.ox.ac.uk/publications/98342
Rapid Generation of Hypomorphic Mutations : IGTRCN
A couple of additional observations. First, it was not poly lysine per se that was causing reduced protein levels because poly AAG (also lysine) did not have the same effects. Second, the strength of the promoter was not important; the poly A track-effect was independent of the strength of the promoter. Finally, the homopolymeric nature of these tracks of A's can be unstable due to the hypermutability of short tandem repeats.. While creating hypomorphic alleles was the authors' primary intent, what they describe is of significance to those working with transgenes. Whereas most who are seeking to express transgenes in insects have been largely limited to the strength of the promoters to manage levels of transgenic proteins, now they can consider modulating transgene using polyA tracks. This could be a very valuable tool in the toolbox of those genetically modifying insects.. Rapid generation of hypomorphic mutations. Laura L. Arthur, Joyce J. Chung, […], Sergej Djuranovic. ...http://igtrcn.org/rapid-generation-of-hypomorphic-mutations/
Vaccine Therapy With gp100 and/or Sargramostim in Treating Patients With Malignant Melanoma - Full Text View - ClinicalTrials...
OBJECTIVES: I. Determine the safety and toxicity of in vivo particle bombardment with DNA coated gold beads carrying cDNA for gp100, with or without gold beads carrying cDNA for sargramostim (GM-CSF), into uninvolved skin of patients with melanoma. II. Estimate the intensity and duration of gp100 transgene expression following these regimens in these patients. III. Assess local lymphocyte phenotype and systemic lymphocyte function following these regimens in these patients. IV. Compare gp100 transgene expression as well as local lymphocyte phenotype and systemic lymphocyte function when the cDNA for GM-CSF is administered 3 days before cDNA for gp100 vs on the same day as gp100 administration in these patients. V. Determine the effect of these regimens on tumor shrinkage, histological evidence of tumor inflammation or necrosis, or in vitro evidence of antitumor immune reactivity in these patients.. OUTLINE: This is a dose escalation study. Patients are assigned to one of three treatment groups. ...https://clinicaltrials.gov/ct2/show/NCT00003897
Chromosomal integration of transgene arrays
General Guidelines Procedure There is a detailed description of the rationale and strategy for making integrants written by Michael Koelle, and there is a thorough discussion of worm transformation in Mello and Fire (1995) Methods in Cell Biology 48, Chapter 19 (pp.451-482). Aside from the oligonucleotide protocol (see below), obtaining an integrant starts with a transmitting line which is treated with a DNA-damaging agent to induce integration. To make the screen efficient and recover multiple independent lines, many researchers use the following procedure.. Briefly, a transmitting line with a moderate precentage of transmission, around 20%, is convenient to use. Approximately 50 late L4 or young adult worms are treated. These animals are separated into groups to allow later assurance of independent lines. To screen for integrants, a two-step approach is used. First, approximately 250 F1 animals are singled out. The progeny are screened for broods which demonstrate >75% transgenic progeny. ...http://www.faculty.ucr.edu/~mmaduro/int.html
Extrapolating empirical results in time and space: modelling tool boxes for predicting introgression of transgenes - NERC...
Hooftman, Danny. 2011 Extrapolating empirical results in time and space: modelling tool boxes for predicting introgression of transgenes. [Keynote] In: Transgenes going wild: Risk assessment of transgene introgression from crops into wild relatives, Leiden, The Netherlands, 11-15 July 2011. (Unpublished) Full text not available from this repository ...http://nora.nerc.ac.uk/503189/
Sense Transgene-induced Silencing is Impaired in dcl2 M | Open-i
Sense Transgene-induced Silencing is Impaired in dcl2 Mutant Plants, but Enhanced in dcl4 Mutants.(A) The diagram shows the coding region of the silenced GUS sehttps://openi.nlm.nih.gov/detailedresult.php?img=PMC2262140_pone.0001755.g001&req=4
Central Metabolism | Max Planck Institute of Molecular Plant Physiology
These intriguing results compelled us to develop a wide range of analytical tools to better study the intricacies of cellular biosynthetic machinery. We have perfected non-aqueous subcellular fractionation techniques in order to separate chloroplasts and vacuoles from cytosol. We are operating a metabolite profiling system, using GC-MS, which allows us to distinguish among large numbers of metabolites within each of these samples (subcellular fractions or tissue samples). In excess of 300 compounds can be profiled in this way , 100 of these compounds having known chemical structures. A further experimental development that we are exploring is the use of chemically-inducible promoters to drive transgene expression in a controlled manner in order to study perturbations of metabolism on a temporal basis. In recent years we have additionally established an RT-PCR platform for tomato transcription factors and sensitive methods for following the metabolism of stable isotope labeled substrate and an ...http://www.mpimp-golm.mpg.de/5858/4fernie
Institute of Cancer Research Repository - Cancer gene therapy: Part 2. Candidate transgenes and their clinical development
Harrington, K. J., Melcher, A. A., Bateman, A. R., Ahmed, A., Vile, R. G. (2002) Cancer gene therapy: Part 2. Candidate transgenes and their clinical development. CLINICAL ONCOLOGY, 14 (2). pp. 148-169. ISSN 0936-6555 ...http://publications.icr.ac.uk/1064/
Gene silencing is a naturally occurring process by which genes can become shut off within a plant. When transgenes are introduced into plants they can also show gene silencing. Genes which share sequence similarity are said to be homologous. When transgenes showing homology to normal cellular genes are introduced they can show a special form of silencing called homology-dependent gene silencing and this typically results in the silencing of one or both genes ...http://www.innovations-report.com/html/reports/life-sciences/report-8999.html
In Vivo Imaging of an Inducible Oncogenic Tumor Antigen Visualizes Tumor Progression and Predicts CTL Tolerance | The Journal...
We described Tg mice, which express a transforming fusion protein, consisting of SV40 large T Ag and luciferase, after site-specific Cre-LoxP recombination. This strategy allowed direct visualization of oncogene expression and noninvasive imaging of tumor growth in a spatiotemporal manner. Surprisingly, in mice of all LoxP-TagLuc-pA founder lines, a ubiquitous and Cre recombination-independent expression of the TagLuc gene was detected by BLI. This leakiness might have resulted from a bicistronic mRNA due to reading through the polyA signal 3′ of the stop cassette. Consequently, CTLs in LoxP-TagLuc-pA mice were tolerant toward the dominant Tag epitope. Because BLI detected Cre recombination-independent TagLuc expression in the thymus, T cell deletion by central tolerance is likely (34). LoxP-TagLuc mice that lacked the polyA signal sequence 3′ of the TagLuc gene showed on average a 10-fold lower Cre recombination-independent TagLuc expression. Because the two transgene DNA constructs ...http://www.jimmunol.org/content/184/6/2930.long
CRISPR with Independent Transgenes or Mutagenic Chain Reaction for Creating Homozygotes in Lab? : IGTRCN
Recently, Port et al. (2015) demonstrated the effectiveness of a less risky method for rapidly producing homozygous-mutant flies. Called CRISPR with Independent Transgenes (CRISPR-it), this method crosses a genomically integrated cas9 expression line with an independent gRNA expression line to target a third genomic site for double-stranded break. Port and colleagues tested several existing cas9 lines to determine their efficiencies for germline and somatic mutations. Although the authors report significant variation between the different cas9 lines (presumably due to genomic position effects), they demonstrated the consistency of their results within any given cas9 line with multiple, independent guides targeting several genes, including some that are homozygous lethal.. Further, Port et al. compared the efficiency of HDR under three conditions: 1) integrated cas9 and integrated gRNA, 2) integrated cas9 with injected guide plasmid, and 3) injection of plasmid expressing both cas9 and the ...https://igtrcn.org/crispr-with-independent-transgenes-or-mutagenic-chain-reaction-for-creating-homozygotes-in-lab/
Janelia Research Campus
Researchers now at Janelia generated over 18,000 driver lines (the VT collection) that exploit the GAL4, LexA, and split-GAL4 systems to express transgenes in distinct and highly ...https://www.janelia.org/open-science/search?f%5B0%5D=search_api_combined_1%3A43410
Persistence and expression of transgenes following in u | Open-i
Persistence and expression of transgenes following in utero rAAV2 gene therapy. In utero gene therapy withrAAV2CMVgfp was performed in NHP and the tissues analyhttps://openi.nlm.nih.gov/detailedresult.php?img=PMC239997_1472-6750-3-16-4&req=4