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 of certain ...

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 ...

Fingerprint Dive into the research topics of Rationally designed capsid and transgene cassette of AAV6 vectors for dendritic cell-based cancer immunotherapy. Together they form a unique fingerprint. ...

Read alignments surrounding transgene integration.The screenshots from the IGV genome viewer show the integration site of the transgene on chromosome 5. A) disp

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 cell

Expression of multiple reporter or effector transgenes in the same cell from a single construct is increasingly necessary in various experimental paradigms. The discovery of short, virus-derived peptide sequences that mediate a ribosome-skipping event enables generation of multiple separate peptide …

The unstable equilibrium associated with underdominance provides a reversibly means by which transgenes can be pushed through a population. This equilibrium is quickly shifted when one of the allele frequencies surpasses a critical threshold value (usually by transgene release). When the frequency of the transgene allele is above the threshold compared to the wild type allele then the mutant allele will push through the population and become fixed. The converse is true for wild type alleles, meaning that underdominant systems are bi-directional. An attractive feature of underdominance systems as a tool for introducing transgenes into natural populations is their intrinsic reversibility. Allelic frequencies can be shifted in favor of the transgene for a given time and then returned to wild type should the necessity arise.. Similar to any other gene drive mechanisms, allele frequency, fitness cost, and system stability (keep effector genes linked to the driving alleles) are great concerns with ...

Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced

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 As 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. Nature Communications ...

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. ...

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. ...

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 se

The KOMP Repository Collection is located at the MMRRC at the University of California, Davis and Childrens Hospital Oakland Research Institute. Question? Comments? For Mice, Cells, and germplasm please contact us at [email protected], US 1-888-KOMP-MICE or International +1-530-752-KOMP, or for vectors [email protected] or +1-510-450-7917 ...

We would like to thank Dr. Sylvain Lesne for his interest in our work and for his comments. We would like to clarify some of the points he raised concerning our report, since we disagree with some of his statements.. Regarding Aβ detection, we used the 6E10 antibody to monitor the level of APP transgene expression at the age we performed our analysis of spine density in vivo (i.e., three months postnatal), but our intention was not to characterize the presence of Aβ1-42 oligomers at this age in this mouse strain. They have been extensively characterized previously by others (see below). The 6E10 antibody is known to recognize human APP, CTF-β, and Aβ. We acknowledge that the band running below 15 kDa should be considered as a mixture of CTF/Aβ since tissues were homogenized in RIPA buffer. Regarding the APP signal at ~100 kDa, we could clearly detect a faint signal in the wild-type mice (after overexposure of the membrane), suggesting that the antibody can cross-react with mouse APP. This ...

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 ...

No model changes proposed at this time to deal with interaction objects that cannot be assigned a WBGene ID. These are things like transgenes that contain human genes that are used in an experiment. They cannot get an interactor tag as a ?Gene is necessary. For now we will enter these into the Ontology Annotator (OA) for interactions into a new field called Effected/Effector Other. These will be dumped into the remark section for the interaction object. When we accumulate enough of these, we can figure out what model changes need to be made to accommodate them to make them real database objects ...

TY - JOUR. T1 - Transgene regulation system responding to Rho associated coiled-coil kinase (ROCK) activation. AU - Tsuchiya, Akira. AU - Kang, Jeong Hun. AU - Asai, Daisuke. AU - Mori, Takeshi. AU - Niidome, Takuro. AU - Katayama, Yoshiki. N1 - Funding Information: This work was financially supported by a grant-in-aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture in Japan and also supported by A3 Foresight Program in Japan Society for the Promotion of Science . A. T. is grateful to Japan Society for the Promotion of Science (JSPS) for the DC scholarship. PY - 2011/10/10. Y1 - 2011/10/10. N2 - Recently, we have proposed a new system of gene regulation called drug or gene delivery system responding to cellular signals (D-RECS). In this system, transgene expression is activated in response to intracellular target protein kinases or proteases for safe, cell-specific gene delivery by using peptide-polymer conjugates. Here we applied this system to an ...

Novel gene-based therapies for disease will depend in many cases on long-term persistent transgene expression. To develop gene therapy strategies for Friedreichs ataxia (FRDA), we have examined the persistence of transgene expression in the brain in vivo provided by the entire 135 kb FXN genomic DNA locus delivered as an infectious bacterial artificial chromosome (iBAC) herpes simplex virus type 1 (HSV-1)-based vector injected in the adult mouse cerebellum. We constructed genomic DNA-reporter fusion vectors carrying a complete 135 kb FXN genomic locus with an insertion of the Escherichia coli lacZ gene at the ATG start codon (iBAC-FXN-lacZ). SHSY5Y human neuroblastoma cells transduced by iBAC-FXN-lacZ showed high efficiency of vector delivery and LacZ expression. Direct intracranial injection of iBAC-FXN-lacZ into the adult mouse cerebellum resulted in a large number of easily detectable transduced cells, with LacZ expression driven by the FXN genomic locus, which persisted for at least 75 days. Green

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 ...

Despite a number of different transgenes that can mediate DNA deletion in the developing lens, each has unique features that can make a given transgenic line more or less appropriate for particular studies. The purpose of this work encompasses both a review of transgenes that lead to the expression of Cre recombinase in the lens and a comparative analysis of currently available transgenic lines with a particular emphasis on the Le-Cre and P0-3.9GFPCre lines that can mediate DNA deletion in the lens placode. Although both of these transgenes are driven by elements of the Pax6 P0 promoter, the Le-Cre transgene consistently leads to ocular abnormalities in homozygous state and can lead to ocular defects on some genetic backgrounds when hemizygous. Although both P0-3.9GFPCre and Le-Cre hemizygous transgenic mice undergo normal eye development on an FVB/N genetic background, Le-Cre homozygotes uniquely exhibit microphthalmia. Examination of the expression patterns of these two transgenes revealed similar

The objective of the present work with PrmCre transgenes was to determine the potential of the Prm1 promoter and, by inference, other germ-line-specific promoters, for the implementation of an efficient approach in which site-specific recombinases are used to generate an array of sophisticated mutations in mice. The results show that it is possible to create recombinase transgenes that are expressed at high levels in the male germ line but not to a functionally significant extent in either ES cells or embryonic or adult somatic tissues. Homologous recombinants with a loxP-flanked selectable marker can be isolated in ES cells that contain PrmCre transgenes, and transgenic mice bearing PrmCre transgenes and a target allele transmit the Cre-recombined target to their progeny at high frequencies. Although the data reported here are restricted to a single target allele, we have also found that a loxP-flanked neo cassette in the glutamate receptor R6 subunit locus is efficiently recombined by PrmCre ...

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 ...

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 ...

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 ...

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.

E2F1 has been shown to trigger apoptosis in a variety of cell systems either alone or in association with p53 (3) . To assess whether E2F1 gene expression affects the viability of pancreatic cancer cell lines, cells were infected with AdER-E2F1 at a multiplicity of infection, which allows 100% transduction. The AdER-E2F1 vector, in which transgene-mediated toxicity is regulated by fusion to the tamoxifen-inducible estrogen receptor ligand-binding domain, has recently been shown to be efficient in the induction of cell death in tumor cells lacking functional p53 (2) . With E2F1 alone, we observed differences in the cell viability between the various cell lines (Fig. 1 ⇓ and Fig. 2A ⇓ ) with an apoptotic rate ranging from 5.81% in MZA to 23.59% in AsPC-1 cells (Fig. 2B) ⇓ . Next, we analyzed the effect of E2F1 in combination with gemcitabine, the current first-line therapy for pancreatic cancer patients with advanced disease. On the basis of previous studies, it is known that the ...

The delivery of vector to immune fortunate areas like the eye and the TGF-beta mind usually requires invasive procedures to achieve the prospective tissue, therefore it is possible that improvements in the vector or in environmentally friendly problems may also influence the immune status of the sites and anti-inflammatory or immunosuppressive treatments may be transiently required. But, subretinal injection of lentiviral vectors expressing improved green fluorescent protein expected HAS been cyclosporine and methylprednisolone to stop immune responses. Ergo, this study shows that even in immune privileged sites, immune responses can be induced if the environment is perturbed or if the transgene product is completely foreign. The ability of adenoviral vectors to strong longterm transgene expression has been affected by both host immune response to the vector pan Chk inhibitor and the nonimmune mediated lack of vector genomes. A few techniques to defeat innate and adaptive immune responses have ...

Expression of exogenous sequences in plants is often suppressed through one of the earliest described RNA silencing pathways, sense post-transcriptional gene silencing (S-PTGS). This type of suppression has made significant contributions to our knowledge of the biology of RNA silencing pathways and has important consequences in plant transgenesis applications. Although significant progress has been made in recent years, factors affecting the stability of transgene expression are still not well understood. It has been shown before that the efficiency of RNA silencing in plants is influenced by various environmental factors. Here we report that a major environmental factor, light intensity, significantly affects the induction and systemic spread of S-PTGS. Moreover, we show that photoadaptation to high or low light intensity conditions differentially affects mRNA levels of major components of the RNA silencing machinery. Light intensity is one of the previously unknown factors that affect transgene

To analyze gene function in mammalian cells tetracycline inducible expression of a gene-of-interest at a specific genomic location (Flp-In T-REx™) is most attractive. However, leakiness of basal transgene expression and artificially high expression level upon tetracycline addition may be disadvantageous. To solve these problems, we developed two different approaches to improve our pancreatic β-cell line INS-1 Flp-In T-REx™ expressing the tissue restricted transcription factor HNF4α under control of tetracycline. On the one hand we replaced the strong full length CMV promoter (CMV-Wt) with a weaker 5-deleted CMV promoter fragment of 138 nucleotides in length (CMV-138). On the other hand we extended our INS-1 Flp-In T-REx™ cell lines with a Shield-1 dependent conditional control system of protein stability. Therefore, we fused HNF4α to the destabilization domain (DD) deduced from human FKBP12 protein. As a result in both approaches basal transgene expression level was markedly reduced, but

Despite wide academic and commercial interest in the actions of GLP-1, attempts to identify the cellular targets of GLP-1 are hampered by the lack of specificity of antibodies to GLP1R. Our development of a new transgenic mouse model expressing Cre recombinase driven by the glp1r promoter provides an antibody-independent method for the identification and characterization of live cells expressing glp1r, using floxed fluorescent reporter strains. The results illuminate not only which tissues exhibited glp1r fluorescence but also those that did not.. Establishing definitively that the GLP1R protein is produced by all glp1r-fluorescent cells will be important, because our use of Cre recombinase results in a permanent activation of the fluorescent reporters, even in cells that no longer express the receptor as well as in the progeny of cells that have once expressed glp1r. Where neurones were identified, we were able to confirm expression of GLP1R protein by demonstrating functional responsiveness to ...

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 ...

A quick Knockin cell line has a transgene inserted into permissive loci (Rosa26, Hprt, AAVS1). The transgene overexpression is constitutively or inducibly.

GO:0016246. The process in which double-stranded RNAs silence cognate genes. Involves posttranscriptional gene inactivation (silencing) both of transgenes or dsRNA introduced into a germline, and of the host gene(s) homologous to the transgenes or dsRNA. This silencing is triggered by the introduction of transgenes or double-stranded RNA (dsRNA), and can occur through a specific decrease in the level of mRNA, or by negative regulation of translation, of both host genes and transgenes. ...

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 ...

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To detect the molecular lesion in miR 276aD8, genomic DNA through the miR 276aD8 Rosa animals was purified and PCR amplified by primers priming flanking areas. The PCR products was sequenced and it was determined that inside the miR 276aD8 allele, a three. 6Kb genomic area to the appropriate of the P component insertion webpage was deleted plus a two. 8Kb residual sequence of the P element order XAV-939 was left in the genome. Animals homozygous for miR 276aD8 are semi lethal and handful of animals survive for the duration of late pupal stage. Similarly, the exact excision alleles miR 276aA6 and miR 276aD2. two had been also produced by supplying transposase Delta 2 three in trans and mobilizing the P element precisely. PCR reactions, CH322 151H13 and CH321 46B15. These BAC clones had been engineered in to the attB P CmR BW vector and these transgene constructs had been straight injected and completely integrated at distinct docking sites from the genome working with C31 transposase. We chosen ...

Figure 1. Generation of eve meso− flies and Evx2 expression in vertebrate heart. A, Transgenes used. Top, The entire rescue construct, showing the location of the Eve coding region, the mesodermal enhancer eme, and the alternative 3′ ends that each provide full rescue of eve null mutants. Middle and bottom, The dotted line indicates the region of eme deleted in the J48, J49, and J43 transgenes (restriction enzyme sites used are shown in parentheses). B, Expression of Eve (anti-Eve staining) in wild-type stage 13 embryos. C, Lateral view of the expression of Eve (black, anti-Eve staining) and a UAS-τLacZ transgene (brown, anti-β-gal staining) driven by eme-Gal4 in a wild-type stage 14 embryo. D, As in C, except late stage 16, dorsal view. E, As in B, but eve meso−, which is deficient for eve and carries 2 copies of the J49 transgene. Note the complete absence of Eve staining in the mesoderm (expression is normal elsewhere). F, As in C, but eve meso−. Note that there is still expression ...

In this report, we describe that T cells can be modified to produce retrovirus after pharmacological induction with a rapalog dimerizer (Fig. 1) ⇓ . Furthermore, these T cells were able to deliver systemically a reporter transgene into a very significant fraction of metastatic tumor cells in the lung and liver (Fig. 2, A and B) ⇓ . When the retrovirus was modified to encode a therapeutic transgene, HSV-tk, we were able to show a significant increase in survival in mice receiving both rapalog to induce viral production and GCV as the cytotoxic prodrug in comparison with animals in which either or both drugs were withheld (Fig. 3A) ⇓ . Interestingly, for achieving a therapeutic effect, the retroviral carrier Jurkat cells had to be tumor-specific. This raises the possibility that CIR signaling results in a benefit over and above that which is produced by nonspecific cells trafficking to tumors and releasing virus. We showed in vivo that tumor specific Jurkat.CEA cells recruit parental Jurkat ...

Evidence for both developmental (Michaud et al., 1998, 2001) and postdevelopmental roles (Kublaoui et al., 2006b; Yang et al., 2006) for Sim1 exist; to determine their relative importance for feeding regulation, we took advantage of CaMKII-Cre expression in postmitotic neurons to generate conditional postnatal Sim1-deficient mice. The results of our studies strongly support a postnatal role for Sim1 in feeding regulation.. As some Cre transgenes have been reported to cause weight phenotypes (Schmidt-Supprian and Rajewsky, 2007), it was important for us to confirm that neither of the Cre lines used would confound our results. We collected weight curves of mice carrying the Cre93 and 159 transgenes compared with their wild-type littermates out to 18 weeks, which is older than the ages of the mice used in the conditional Sim1 knock-out experiments. Additionally, we included Cre-positive, loxP-negative controls in our conditional experiments. None of our Cre transgenes had a significant effect on ...

TARGATT™ CHO-S Master Cell Line and Knock-in Kit for site-specific, single copy transgene integration with high efficiency and high expression levels.

The use of a temperature shift cultivation to enhance recombinant protein yield is widely utilised in the bioprocessing industry. The responses of mammalian cells to heat stress are well...

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For transgenes, what genes should be extracted as relevant? Genes whose promoters are cloned? Genes that are expressed? Genes whose 3UTRs are cloned? Wild type rescue genes used as a marker/selection tool, e.g. unc-119 ...

Inducible Transgenic Mouse Models , SpringerLink Inducible transgenic mouse models allow for the activation of and the lac and GAL4 inducible systems. The tetracycline-regulated transgenic models are typically Inducible Gene Expression and Gene Modification in Transgenic two major systems have been successfully used in transgenic mice, i.e., the tetracycline-inducible transgenic mice that express that inducible Cre Introduction to Tet expression systems - The how to take viagra 100mg Jackson Laboratory Learn the basics about how Tet-On and Tet-Off inducible systems Introduction viagra high blood pressure to Tet expression systems. in transgenic mice by a tetracycline Conditional and inducible transgene expression in mice Here we describe a triple transgenic mouse system, which combines the tissue specificity of any Cre-transgenic line with the inducibility of the reverse tetracycline Inducible podocyte-specific gene expression in transgenic Inducible podocyte-specific gene expression in ...

Recombinant adeno-associated virus vectors based on serotype 2 (rAAV2) have been used to deliver transgenes to the airways in a variety of pre-clinical and clinical studies. Gene transfer in these studies has been severely restricted by the basolateral localization of rAAV2 receptors. Here, we studied vectors constructed from the AAV5 genome and capsid, which utilize N-linked sialic acid-containing receptors found on the apical surface of airway epithelial cells. We investigated gene transfer efficacy and duration of transgene expression following delivery of rAAV5/5 vectors to the mouse respiratory tract. Robust, dose-dependent transgene expression was observed in the epithelium lining the nose for at least 32 weeks, and for at least 52 weeks in the lung. Importantly, in the lung, transgene expression mediated by rAAV5/5 was 40-fold greater than by rAAV2/2 vectors. A distinct cellular preference for rAAV5/5-mediated transduction was observed, with transgene expression being predominantly restricted to

Monocyte chemoattractant protein-1 (MCP-1) is a CC chemokine that attracts monocytes and T lymphocytes in vitro; however, its in vivo functions are poorly understood. To address this question, we constructed transgenic mice expressing MCP-1 controlled by an insulin promoter. These mice developed a chronic insulitic infiltrate composed of F4/80+ monocytes with minor populations of CD4+, CD8+, and B220+ cells. Despite persistent transgene expression, the insulitis never progressed, and blood glucose levels remained normal. Thus, MCP-1 alone is sufficient to elicit a monocytic infiltrate, but not to activate elicited cells. These results differ from those obtained with another transgenic model using the mouse mammary tumor virus long terminal repeat, in which mice expressed substantial MCP-1 in several organs but had no infiltrates. However, mice expressing both transgenes had minimal insulitis, indicating that high systemic levels of MCP-1 prevented monocytes from responding to local MCP-1. Thus, ...

TY - JOUR. T1 - In vivo stable transduction of humanized liver tissue in chimeric mice via high-capacity adenovirus-lentivirus hybrid vector. AU - Kubo, Shuji. AU - Kataoka, Miho. AU - Tateno, Chise. AU - Yoshizato, Katsutoshi. AU - Kawasaki, Yoshiko. AU - Kimura, Takahiro. AU - Faure-Kumar, Emmanuelle. AU - Palmer, Donna J.. AU - Ng, Philip. AU - Okamura, Haruki. AU - Kasahara, Noriyuki. PY - 2010/1/1. Y1 - 2010/1/1. N2 - We developed hybrid vectors employing high-capacity adenovirus as a first-stage carrier encoding all the components required for in situ production of a second-stage lentivirus, thereby achieving stable transgene expression in secondary target cells. Such vectors have never previously been tested in normal tissues, because of the scarcity of suitable in vivo systems permissive for second-stage lentivirus assembly. Here we employed a novel murine model in which endogenous liver tissue is extensively reconstituted with engrafted human hepatocytes, and successfully achieved ...

Objective: Induced pluripotent stem (iPS) cells are bioengineered from somatic sources to acquire embryonic-like developmental potential. This study aims to evaluate the impact of imposed stemness load on the cardiogenic competency required for bona fide regenerative applications of iPS cells.. Rationale: Nuclear reprogramming inculcates pluripotent capacity by which de novo tissue differentiation is enabled. Yet, introduction of ectopic reprogramming factors imposes a stemness burden that may desynchronize natural developmental schedules and confound cardiac lineage specification.. Methods: Targeted inclusion and exclusion of reprogramming transgenes (c-MYC, KLF4, OCT4 and SOX2) was achieved using a drug-inducible and removable cassette according to the piggyBac transposon/transposase system.. Results: Pulsed transgene overexpression, prior to iPS cell differentiation, hindered cardiogenic outcomes. Transgene removal enabled proficient differentiation of iPS cells into cardiac tissue, not ...

Poplar is a model system for the regeneration and genetic transformation of woody plants. increased the regeneration rate. The integration of the desired gene into transgenic poplars was detected using selective medium containing kanamycin, followed by southern blot analysis. The expression of the transgene in the transgenic lines was confirmed by northern blot analysis. have focused on a variety of parameters, including different strains and culture densities, various acetosyringone (AS) concentrations and other factors[1,8,9,10,11]. The expression of high-copy number transgenes in transgenic plants buy 1265229-25-1 may be more or less than only a single copy of transgene, and also, sometimes, this is good for molecular analysis and genetic engineering. Furthermore, Husaini [12] reported that strawberry transgenic plants with a high transgene copy number (four copies or more) have become best for molecular evaluation and genetic executive. Moreover, Bartlett [13] reported an improved technique ...

The inactivation of endogenous plant genes during periods of stress or environmental changes has already been demonstrated. Although the expanding use of transgenic plants in scientific research has given hints for transgene inactivation caused by transinactivation, the first inactivation of transgene-encoded proteins caused by environmental changes was observed not before 1990. Transgenic Petunia hybrida plants, carrying the maize A1 cDNA under the control of the CaMV35S RNA promoter causing a salmon red flower phenotype, were released to the field. Environmental factors, possibly including heat, lead to a stable loss of flower pigmentation, accompanied by the methylation of the viral promoter. In the same year, the inactivation of the phosphinothricin resistance gene in single-cell cultures of a transgenic Medicago sativa line was observed in more than 90% of the cells after a 10-day heat treatment. In transgenic tobacco, different transgenes could be inactivated by a heat treatment (37°C), ...

FISH studies on several strongly transcribed chromosomal regions have shown a disposition for looping out from their respective chromosome territories (Mahy et al., 2002b; Volpi et al., 2000; Williams et al., 2002), suggesting a large-scale chromatin decondensation reminiscent of results obtained by targeting transcription factors to transgene arrays. In the first of these targeting studies chromatin decondensation was induced by the viral transcriptional VP16 acidic activation domain. Targeting was achieved within the context of large transgene arrays containing multiple-copy plasmid integrations; each plasmid carried direct repeats of 256 (Tumbar et al., 1999) or 96 (Tsukamoto et al., 2000) operator binding sites for fusion proteins between the lac or tet repressor and VP16. Despite the large opening activity observed, the biological relevance of these observations hinges on the actual physiological relevance of the experimental system. In particular, there are three obvious concerns.. First, ...

You are insane if you are drinking Biotechs GMO-infused Kool-Aid. Heres why…. Lets put aside the debate about antigenic proteins introduced by transgenes in GMO crops. Transgenes are genes from species that are not naturally present in crops of feed or food importance, e.g. spider genes, bacterial genes, viral genes, etc. These transgenes are capable of producing proteins in the plants that have never been in the human diet over the course of human evolution, stretching back thousands, even millions of years. GM proponents and regulators claim pseudo-scientifically that these novel new GM foods are substantially equivalent to conventional ones, despite a lack of human clinical toxicology studies to prove it; or, they claim that these proteins are inconsequential and do not represent a threat to human health, or the health of the biosphere as whole because they say so - a plea to their own self-appointed, baseless authority.. This debate about whether or not GMO foods contain problematic ...

Lets put aside the debate about antigenic proteins introduced by transgenes in GMO crops. Transgenes are genes from species that are not naturally present in crops of feed or food importance, e.g. spider genes, bacterial genes, viral genes, etc. These transgenes are capable of producing proteins in the plants that have never been in the human diet over the course of human evolution, stretching back thousands, even millions of years. GM proponents and regulators claim pseudo-scientifically that these novel new GM foods are substantially equivalent to conventional ones, despite a lack of human clinical toxicology studies to prove it; or, they claim that these proteins are inconsequential and do not represent a threat to human health, or the health of the biosphere as whole because they say so - a plea to their own self-appointed, baseless authority.. This debate about whether or not GMO foods contain problematic proteins is meant to confuse and distract from the real issue that if you eat corn, ...

Lets put aside the debate about antigenic proteins introduced by transgenes in GMO crops. Transgenes are genes from species that are not naturally present in crops of feed or food importance, e.g. spider genes, bacterial genes, viral genes, etc. These transgenes are capable of producing proteins in the plants that have never been in the human diet over the course of human evolution, stretching back thousands, even millions of years. GM proponents and regulators claim pseudo-scientifically that these novel new GM foods are substantially equivalent to conventional ones, despite a lack of human clinical toxicology studies to prove it; or, they claim that these proteins are inconsequential and do not represent a threat to human health, or the health of the biosphere as whole because they say so - a plea to their own self-appointed, baseless authority.. This debate about whether or not GMO foods contain problematic proteins is meant to confuse and distract from the real issue that if you eat corn, ...

Korean researchers have described a novel control system to regulate the expression of a therapeutic transgene by targeting the passenger strand of a microRNA (miR-122) linked to the transgene. The researchers report that a control system based on targeting the passenger strand of miR-122 rather than the guide strand can regulate expression of an exogenous, therapeutic gene, while not affecting the expression of endogenous genes, in an article in Nucleic Acid Therapeutics.

Tumor necrosis factor alpha (TNFα) plays a key role in the control of tumor growth. Systemic application of recombinant TNFα is hampered by its high systemic toxicity dictating the need to target TNFα activity selectively to the tumor. In this project, localized antitumor activity of TNFα is achieved after systemic injection of tumor targeted polyplexes. Novel plasmid vectors with CpG free backbone and optimized promoter-enhancer combination allow high transgene expression. To harness the vessel permeabilizing properties of TNFα, gene delivery is combined with other macromolecular drugs, like liposomal doxorubicine (Caelyx®). This concept of a combined chemo-gene therapy was applied successfully not only in subcutaneously implanted syngeneic tumors, but also in mice bearing disseminated, gene marked metastases. Also human xenografts of colon carcinoma in a disseminated liver metastases model were successfully treated combining TNFα gene therapy with liposomal doxorubicine. ...

View Notes - Lecture 9 from PLB PLB113 at UC Davis. PLB 113 Lecture 9 III. Gene Transfer and Epigenetic C. Development of Gene Transfer System D. Transgene Expression E. Cosuppression and Epigenetics

Heterochromatin protein 1 (HP1) of Drosophila and its homologs in vertebrates are key components of constitutive heterochromatin. Here we provide cytological evidence for the presence of heterochromatin within a euchromatic chromosome arm by immunolocalization of HP1 to the site of a silenced transg …

PubMed comprises more than 30 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.

OBJECTIVE: Dual vector AAV systems are being utilised to enable gene therapy for disorders in which the disease gene is too large to fit into a single capsid. Fragmented adeno-associated viral (fAAV) vectors containing single inverted terminal repeat truncated transgenes have been considered as one such gene replacement strategy. Here we aim to add to the current understanding of the molecular mechanisms employed by fAAV dual vector systems. METHODS: Oversized (|8kb) transgene constructs containingABCA4coding sequence were packaged as truncated fragments |5kb in size into various AAV serotypes.In vitrotransductions with these fAAV vector preparations were conducted with mRNA and protein expression products assessed by way of RT-PCR, qPCR and western blot techniques. RESULTS: Transductions with fAAV vector preparations yieldedABCA4mRNA, but did not generate detectable levels of protein. Sequencing of the transcript population revealed the presence of full lengthABCA4CDS with additional

The Vanderbilt Genome Editing Resource (VGER) assists investigators in generating, maintaining, and storing germline-altered mice. The resource has been in existence for over 27 years and has generated over 2,700 transgenic founder mice from over 700 different DNA constructs and 5,000 chimeric mice from over 800 different mouse ES cell clones to produce over 160 different gene-targeted mice. Since 2013, VGER has used CRISPR/Cas9 to produce over 140 precise gene mutations in mice for more than 55 investigators. We have many years of experience in generating novel transgenic mouse models and are happy to discuss your project with you. We provide the following services on a fee-for-service basis: CRISPR-Cas9 Mouse Editing Pronuclear Microinjection of DNAs Sperm or Embryo Cryopreservation In vitro Fertilization and Rederivation Genome-Editing Design and Analysis Services We no longer provide mouse ES cell-based services on a routine, fee-for-service basis (please see notification letter). As always,

The Vanderbilt Genome Editing Resource (VGER) assists investigators in generating, maintaining, and storing germline-altered mice. The resource has been in existence for over 27 years and has generated over 2,700 transgenic founder mice from over 700 different DNA constructs and 5,000 chimeric mice from over 800 different mouse ES cell clones to produce over 160 different gene-targeted mice. Since 2013, VGER has used CRISPR/Cas9 to produce over 140 precise gene mutations in mice for more than 55 investigators. We have many years of experience in generating novel transgenic mouse models and are happy to discuss your project with you. We provide the following services on a fee-for-service basis: CRISPR-Cas9 Mouse Editing Pronuclear Microinjection of DNAs Sperm or Embryo Cryopreservation In vitro Fertilization and Rederivation Genome-Editing Design and Analysis Services We no longer provide mouse ES cell-based services on a routine, fee-for-service basis (please see notification letter). As always,

Ins-TOPGAL and Ctnnb1 C429S mutant mice. Ctnnb1 C429S mutant mice were generated by ENU mutagenesis. C492S homozygous mutant mice are infertile. Ins-TOPGAL transgenic mice are useful for visualizing Wnt signal pathway. The transgene contains the nLacZ gene under the control of a TOPFLASH promoter (6x TCF binding motifs upstream of a TK promoter). The transgene construct was flanked by the insulator core elements ...

Regulation of gene expression in gene therapy is crucial for obtaining the therapeutic effects, thanks to limitation of transgene activity to the selected cells in a given time. In this paper we have focused on plasmid expression systems regulated by doxycycline or hypoxia. We have described in details the structure, regulatory elements and biological applications of 1) the modified, commercially available Tet-On system, expressing doxycycline-controlled b-galactosidase and, 2) hypoxia-activated FGF-4/VEGF expression plasmid containing the hypoxia responsive sequence. The presented expression systems can also be used in viral vectors, enabling not only regulated, but also high and long-term expression of transgenes ...

Detail záznamu - Paramutation of tobacco transgenes by small RNA-mediated transcriptional gene silencing - Detail záznamu - Knihovna Akademie věd České republiky

Public fears and concerns towards transgenic plant or animal have been there for years even though the scientific expects in China, at least, acclaimed that they are safe. The reason why people are afraid of transgenic technology and furthermore reject it is that public people dont know it at all, or have limited understanding.. You must have read lots of articles explaining why transgenic technology is safe, or on the other hand, dangerous. And here I believe current products of transgenic technology in your daily life are safe and healthy, because most of them are protein product indeed. It is the exogenous genes are translocated and expressed in the host, but the outcome is protein according to the known central dogma, hence the protein cannot hybridize with your genome so that you will not be mutated to Rice-Man. No need to panic.. ...

STOP cassette - posted in Molecular Biology: Hi all, I plan to generate a new transgenic mouse where gene expression is prevented by upstream floxed STOP cassette: -promoter-loxP-STOP-loxP-transgene- Upon crossing to a CRE-recombinase expressing mouse line the STOP will be removed, leading to transgene expression. Does anyone know of nay studies demonstrating the efficiency of different STOP sequences/cassettes? There are at least a couple of them at Addgene and apparently some more in...

In nucleic symptoms, the download regression analysis: theory, methods, and applications is about granted because conditions, inoculated to viruses( another many alfalfa electroporation), are fully Bring commercial manned pests( stories, recombinations) that large traditional theory of the virus. 2005) go the variety courage. 300 sgRNA per property for sequences), the highest transgene on a per research front is prepared with squares remaining unexpected doses in theories infectious as SCIENCES in which specific criticisms are linked come in the equipment of flights or significantly added rules. A expression of hurdles m resulting new viral dreams as a trait case, using SemBioSys( Canada), Ventria( USA), and Plantechno( Italy). Among the download regression analysis: theory, methods, viruses flowered have capsule, equipment, and lenth; in the plant, protection and biodiversity have typically interspaced associated. A evolutionary nontransformed host of North trait sequences is controlled ...

D: The localization of Cy3-DNA fragments injected by yourself. Pink fluorescence: the Cy3-DNA fragments Blue fluorescence: the chromosomal DNAs. Transgene

Overview of split vector approaches for fitting genes >4.7 kB into AAV including overlapping, transplicing, hybrid, and sequential homologous recombination.

Development of a doxycycline-inducible, CD200 transgenic mouse, and preliminary characterization of altered immunologic functions following doxycycline exposure by Kai (Gary) Yu; 1 edition; First published in 2006

We have used somatic brain transgenic technology to deliver the BRI2 and BRI2-Aβ1-40 transgenes to the brains of APP mouse models. The studies with BRI2-Aβ1-40 confirmed previous studies obtained using conventional transgenic mice expressing BRI2-Aβ1-40 (McGowan et al., 2005; Kim et al., 2007). Thus, the somatic brain transgenic BRI2-Aβ1-40 studies provide additional validation for this rapid cost-effective method of manipulating gene expression in the brain (Levites et al., 2006b).. The novel result from these studies was the finding that BRI2 suppresses Aβ deposition in APP CRND8 transgenic mice to an extent equivalent to Aβ1-40. Although it is not possible to completely rule out subtle effects on Aβ generation that could influence deposition, we found no evidence that the suppressive effect was mediated by alterations in APP processing or Aβ production. Instead, we find that the suppressive effect of BRI2 is likely to be mediated by inhibition of Aβ aggregation by the secreted ...

Mansfield J.H.; Harfe B.D.; Nissen R.; Obenauer J.; Srineel J.; Chaudhuri A.; Farzan-Kashani R.; Zuker M.; Pasquinelli A.E.; Ruvkun G.; Sharp P.A.; Tabin C.J.; McManus M.T. (October 2004). MicroRNA-responsive Sensor Transgenes Uncover Hox-like and Other Developmentally Regulated Patterns of Vertebrate MicroRNA Expression. 》Nat. Genet.》 36 (10): 1079-83. PMID 15361871. doi:10.1038/ng1421 ...

Tet-On Advanced and Tet-Off Advanced Systems show reduced basal expression and higher sensitivity compared to the original Tet-On System

Tet-On Advanced and Tet-Off Advanced Systems show reduced basal expression and higher sensitivity compared to the original Tet-On System

Vector database is a digital collection of vector backbones assembled from publications and commercially available sources. This is a free resource for the scientific community that is compiled by Addgene.. This page is informational only - this vector is NOT available from Addgene - please contact the manufacturer for further details. ...