Variegation in Drosophila is a manifest illustration of the important role played by chromatin structure in gene expression. Mutants of modulo (mod) have been isolated and this gene is shown to be a dominant suppressor of variegation. Null mutants are recessive lethal with a melanotic tumour phenotype. The mod protein directly binds DNA, which indicates that it may serve to anchor multimeric complexes promoting chromatin compaction and silencing (Garzino, 1992). To analyse the consequences of mod loss of function in mitotically active cells of the imaginal disc, clones of cells homozygous for the null allele A4-4L8 were generated by FLP-mediated recombination. In these experiments, clones were identified on the adult epidermis of mosaic animals by the loss of yellow and Stubble bristle markers. mod-deficient clones were found in adult flies on head, thorax, legs and abdomen. They present three main features: (1) they are systematically of reduced size when compared to controls (wild-type clones ...
One of the models in the Taconic Biosciences GEM Collection, Kcnk3 - Model 8734 - cKO: the conditional KO allele has been generated after Flp-mediated recombination by crossing chimeras to a Flp-Deleter on a C57BL/6 background.
Author: Jahnz, M. et al.; Genre: Journal Article; Published in Print: 2002-01; Title: FLP recombinase: Changing substrate specificity by evolutionary biotechnology
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This chapter reviews the biochemical, mechanistic, and topological aspects of the Flp system with special emphasis on the contributions made by the Flp protein subunits and the target DNA to the individual steps of the recombination pathway. It illustrates how Flp recombination fits into the global physicochemical paradigm followed by the integrase/tyrosine family recombinases, while still retaining features that are strikingly different from other members of the family. The chapter then discusses how the Flp active site provides a model for the emergence of complex active sites from elementary active sites under the functional constraints faced by biological catalysts during their evolution. Next, it alludes to some of the applications of Flp in yeast and in extraneous host systems. Researchers conclude by considering potential mechanisms for Flp regulation in vivo to rapidly commission or decommission the amplification machine as demanded by the copy-number status of the 2μm plasmid. The DNA-protein
Hi, I am have trouble with getting stable expression of DsRed in HEK cells. I am using the ,a href=http://www.ncbi.nlm.nih.gov/pubmed/ 11730010, pStoplight vector,/a, which express DsRed until it is cut out by Cre Recombinase, and then GFP is expressed. I have cloned this into the ,a href=http://products.invitrogen.com/ivgn/en/US/adirect/invitrogen? cmd=catProductDetail&entryPoint=adirect&productID=K601001&messageType=catProductDetail&showAddButton=true,Flip- in kit,/a, from Invitrogen to generate a stable cell line using flp recombinase to integrate the vector. It looks good for a while, but then, even though I have cells surviving the selection agent, after a week or so, I dont have any more red fluorescent cells! So, I have no idea whats happening. It seems to me that either: 1) the transient expression of flp recombinase is somehow acting on all the stoplight transfected cells, somehow removing the gene... unlikely. 2) DsRed expression is somehow being driven down by the cell? 3) DsRed ...
Recombinases are genetic recombination enzymes. DNA recombinases are widely used in multicellular organisms to manipulate the structure of genomes, and to control gene expression. These enzymes, derived from bacteria and fungi, catalyze directionally sensitive DNA exchange reactions between short (30-40 nucleotides) target site sequences that are specific to each recombinase. These reactions enable four basic functional modules, excision/insertion, inversion, translocation and cassette exchange, which have been used individually or combined in a wide range of configurations to control gene expression. Types include: Cre recombinase Hin recombinase Tre recombinase FLP recombinase Nern, A; Pfeiffer, BD; Svoboda, K; Rubin, GM (Aug 23, 2011). "Multiple new site-specific recombinases for use in manipulating animal genomes". Proceedings of the National Academy of Sciences of the United States of America. 108 (34): 14198-203. doi:10.1073/pnas.1111704108. PMC 3161616 . PMID 21831835. García-Otín, AL; ...
In Drosophila, P-GAL4 enhancer trap lines can target expression of a cloned gene, under control of a UAS(GAL) element, to any cells of interest, However, additional expression of GAL4 in other cells can produce unwanted lethality or side-effects, particularly when It drives expression of a toxic gene product. To target the toxic gene product ricin A chain specifically to adult neurons, we have superimposed a second layer of regulation on the GAL4 control. We have constructed flies in which an effector gene is separated from UAS(GAL), by a polyadenylation site flanked by two FRT sites in the same orientation; A recombination event between the two FRT sites, catalysed by yeast FLP recombinase, brings the effector gene under control of UAS(GAL). Consequently, expression of tile effector gene is turned on in that cell and its descendants, if they also express GAL4. Recombinase is supplied by heat shock induction of a FLP transgene, allowing both tinting and frequency of recombination events to be ...
... , Authors: Dessen P. Published in: Atlas Genet Cytogenet Oncol Haematol.
Marketer account activation forces gene transcriptional result. effective strategy in probing and understanding the systems of gene transcription gene marked with 24X Master of science2 repeats and analysed its transcriptional account activation kinetics during serum response15. In another strategy, single-copy transgenes powered by the marketer and the marketer had been produced by site-specific DNA recombination in HEK293 individual embryonic kidney cells that allowed analysing transcription kinetics at the single-mRNA level24. non-etheless, provided that the mammalian genome is normally transcribed25 pervasively, this function do not really address to what level transgene reflection shown marketer account activation or mimicked the endogenous gene. In this paper, we survey an fresh program to measure transcriptional result from a single-copy transgene powered by the cell-cycle governed marketer. Cyclins are an essential group of highly-conserved protein that interact with cyclin-dependent ...
Integrase family 250 binary option trading demo develop after therapeutic Australia , pokemon trading card storage, forexpros live charts, maximus forex, car trading com
Site-specific recombination at the plasmid ColE1 cer site requires the Escherichia coli chromosomal gene xerC. The xerC gene has been localized to the 85-min region of the E. coli chromosome, between cya and uvrD. The nucleotide sequences of the xerC gene and flanking regions have been determined. The xerC gene encodes a protein with a calculated molecular mass of 33.8 kDa. This protein has substantial sequence similarity to the lambda integrase family of site-specific recombinases and is probably the cer recombinase. The xerC gene is expressed as part of a multicistronic unit that includes the dapF gene and two other open reading frames. ...
TY - JOUR. T1 - Clinical Utility of Initial Terminal Deoxynucleotidyl Transferase Determinations in Childhood Acute Leukemias. AU - Kalwinsky, David K.. AU - Weatherred, William H.. AU - Dahl, Gary V.. AU - Bowman, W. Paul. AU - Melvin, Susan L.. AU - Coleman, Mary Sue. AU - Bollum, F. J.. PY - 1981/7/1. Y1 - 1981/7/1. N2 - Terminal deoxynucleotidyl transferase (TDT) activity was measured in bone marrow lymphoblasts obtained at diagnosis from 168 consecutive patients with childhood acute leukemia. Absolute concentrations of TDT were increased (,20 units/108 blasts) in samples from 98 of 112 assessable patients with acute lymphocytic leukemia (ALL). The values ranged from ,1 to 1502 units/108 blasts with a median of 90 units contrasted with ,1 to 219 units (median, 2.6 units) in studies of children without leukemia. Results of an immunofluorescence assay were in good agreement with enzymatic detection of the polymerase. Among 115 patients with adequate marrow smears, 105 had TDT-positive blasts. ...
Roychoudhury, R and Bloch, D P., "Studies on deoxyribonucleic acid polymerase from ehrlich ascites tumor cells. II. Factors influencing primer and template requirement of deoxyribonucleic acid polymerase." (1969). Subject Strain Bibliography 1969. 746 ...
The advent of site-specific recombinase (SSR) technology and the Cre/lox system has led to numerous advances in molecular biology, and...
Methods and compositions using populations of randomized modified FRT recombination sites to identify, isolate and/or characterize modified FRT recombination sites are provided. The recombinogenic mod
Animal work. Diabetic db/db, mice and their control littermates db/m were obtained from The Jackson Laboratory (strain: BKS.Cg-Dock7m+/+Leprdb/J) and bred in-house. All animals were maintained on a normal chow diet with free access to water and housed in a room with a 12-hour light/12-hour dark cycle and an ambient temperature of 22°C. Type 1 diabetes was induced in mice as previously described (13). Drp1S600A-knockin mice. We designed a Drp1S600A-knockin construct using a replacement targeting strategy that involved 3 cloning steps. First, a 3.5-kb genomic DNA encompassing exons 15-17 and the proximal intronic regions of the Drp1 gene was PCR amplified and subcloned directly into a pLNTK vector. This DNA fragment was sequencing verified and used as the 5′ homologous recombination arm. Second, a neomycin-resistant minigene cassette flanked by flippase recognition target (FRT) sequences was added to the 3′ end of the 5′ arm in the pLNTK backbone. Third, a 5.1-kb genomic fragment ...
Cre recombinase兔多克隆抗体(ab40011)经WB实验严格验证,被1篇文献引用并得到3个独立的用户反馈。所有产品均提供质保服务,中国75%以上现货。
DNTTIP1 antibody [1F4] (deoxynucleotidyltransferase, terminal, interacting protein 1) for ICC/IF, IHC-P, WB. Anti-DNTTIP1 mAb (GTX84604) is tested in Human samples. 100% Ab-Assurance.
New recombination systems provide refinement of the Cre-lox system, to spatiotemporally regulate gene expression, creating more sophisticated mouse models.
P elements containing a 7 kb DNA fragment from the middle of the Drosophila bithorax complex insert preferentially into the bithorax complex or into the adjacent chromosome regions. This homing property is similar to that reported for the engrailed promoter (Hama, C., Ali, Z. and Kornberg, T. B. (1990) Genes Dev. 4, 1079-1093). The 7 kb fragment does not contain any known promoter, but it acts as a boundary element separating adjacent segmental domains. An enhancer-trap P element was constructed with the homing fragment and the selectable marker flanked by FRT sites. P insertions can be trimmed down by Flp-mediated recombination to just the lacZ reporter, so that the (beta)-galactosidase pattern is not influenced by sequences inside the P element. Twenty insertions into the bithorax complex express (beta)-galactosidase in segmentally limited patterns, reflecting the segmental domains of the bithorax complex where the elements reside. The mapping of segmental domains has now been revised, with ...
Terminal deoxynucleotidyl transferase (TdT), also known as DNA nucleotidylexotransferase (DNTT) or terminal transferase, is a specialized DNA polymerase expressed in immature, pre-B, pre-T lymphoid cells, and acute lymphoblastic leukemia/lymphoma cells. TdT adds N-nucleotides to the V, D, and J exons of the TCR and BCR genes during antibody gene recombination, enabling the phenomenon of junctional diversity. In humans, terminal transferase is encoded by the DNTT gene. As a member of the X family of DNA polymerase enzymes, it works in conjunction with polymerase λ and polymerase μ, both of which belong to the same X family of polymerase enzymes. The diversity introduced by TdT has played an important role in the evolution of the vertebrate immune system, significantly increasing the variety of antigen receptors that a cell is equipped with to fight pathogens. Studies using TdT knockout mice have found drastic reductions (10-fold) in T-cell receptor (TCR) diversity compared with that of normal, ...
Studying the biophysical characteristics of glycosylated proteins and solving their three-dimensional structures requires homogeneous recombinant protein of high quality.We introduce here a new approach to produce glycoproteins in homogenous form with the well-established, glycosylation mutant CHO Lec3.2.8.1 cells. Using preparative cell sorting, stable, high-expressing GFP master cell lines were generated that can be converted fast and reliably by targeted integration via Flp recombinase-mediated cassette exchange (RMCE) to produce any glycoprotein. Small-scale transient transfection of HEK293 cells was used to identify genetically engineered constructs suitable for constructing stable cell lines. Stable cell lines expressing 10 different proteins were established. The system was validated by expression, purification, deglycosylation and crystallization of the heavily glycosylated luminal domains of lysosome-associated membrane proteins (LAMP).
XerC is a site-specific recombinase of the bacteriophage lambda integrase family that is encoded by xerC at 3700 kbp on the genetic map of Escherichia coli. The protein was originally identified through its role in converting multimers of plasmid ColE1 to monomers; only monomers are stably inherited …
The tyrosine family of recombinases produces two smaller DNA circles when acting on circular DNA harboring two recombination sites in head-to-tail orientation. If the substrate is supercoiled, these circles can be unlinked or form multiply linked catenanes. The topological complexity of the products varies strongly even for similar recombination systems. This dependence has been solved here. Our computer simulation of the synapsis showed that the bend angles, phi, created in isolated recombination sites by protein binding before assembly of the full complex, determine the product topology. To verify the validity of this theoretical finding we measured the values of phi for Cre/loxP and Flp/FRT systems. The measurement was based on cyclization of the protein-bound short DNA fragments in solution. Despite the striking similarity of the synapses for these recombinases, action of Cre on head-to-tail target sites produces mainly unlinked circles, while that of Flp yields multiply linked catenanes. In full
Recombination directionality factors (RDFs), or excisionases, are essential players of prophage excisive recombination. Despite the essentially catalytic role of the integrase in both integrative and excisive recombination, RDFs are required to direct the reaction towards excision and to prevent re-integration of the prophage genome when entering a lytic cycle. KplE1, HK620 and numerous (pro)phages that integrate at the same site in enterobacteria genomes (such as the argW tRNA gene) all share a highly conserved recombination module. This module comprises the attL and attR recombination sites and the RDF and integrase genes. The KplE1 RDF was named TorI after its initial identification as a negative regulator of the tor operon. However, it was characterized as an essential factor of excisive recombination. In this study, we designed an extensive random mutagenesis protocol of the torI gene and identified key residues involved in both functions of the TorI protein. We show that, in addition to TorI-TorR
Until recently, a relatively simple model was proposed for the biological function of the amino-terminal domain of the lambda Int. The high-affinity amino-terminal domain binds to the arm-type sequence to deliver the low-affinity core-binding domain of Int to the core-type site, where actual cleavage and rejoining occur (9, 12, 13). Simultaneous binding to the two different DNA sites by a single Int molecule is further facilitated by accessory proteins that bind and bend the sites between the arm-type and the core-type sequences (9, 13).. Several recent studies, however, revealed more elaborate roles of the amino-terminal domain of the Int protein in addition to the architectural role. The amino-terminal domain is implicated as a region involved in protein-protein interactions between Int molecules during intasome formation (8). Some amino acid substitutions in one domain may alter the structure of the other domain through domain-domain communication. A few amino acid substitutions in the HK022 ...
Candida parapsilosis is a major cause of human disease, yet little is known about the pathogens virulence. We have developed an efficient gene deletion system for C. parapsilosis based on the repeated use of the dominant nourseothricin resistance marker (caSAT1) and its subsequent deletion by FLP-mediated, site-specific recombination. Using this technique, we deleted the lipase locus in the C. parapsilosis genome consisting of adjacent genes CpLIP1 and CpLIP2. Additionally we reconstructed the CpLIP2 gene, which restored lipase activity. Lipolytic activity was absent in the null mutants, whereas the WT, heterozygous, and reconstructed mutants showed similar lipase production. Biofilm formation was inhibited with lipase-negative mutants and their growth was significantly reduced in lipid-rich media. The knockout mutants were more efficiently ingested and killed by J774.16 and RAW 264.7 macrophage-like cells. Additionally, the lipase-negative mutants were significantly less virulent in infection ...
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Recombinase-mediated cassette exchange enables the swapping of large genomic regions, and recommended for the generation of humanized models.
... is a special type of site-specific recombination. The Cre protein is a site-specific DNA recombinase. It can catalyze the recombination of DNA between specific sites in a DNA molecule. These sites, known as loxP sequences, contain specific binding sites for Cre that surround a directional core sequence where recombination can occur. It is often used in the generation of knockout and conditional knockout animals. - Cre-Lox Recombination - AbVideo™ - Support - Abnova
Hi all, I was wondering if any has hear of a higher eukaryotic gene organization such as this : 2 genes, intronless, facing towards eachother (promotion-wise) that are absolutly critical for the function of a system. these genes show no homology to eachother (or anyother genes for that matter) but are conserved for over 500 million years of evolution. they are absolutly critical for function. these are the recombinase activating genes. they are essential for immune system function-t and b cell rearrangement. a possible theory is that they are the resylt of horizontal gene transfer from a viral infection (intronless, 2 genes for one function right next to each other on the same chromasome...ect) 500 million years ago or so. they are almost completly conserved over this time. question: can anyone support the viral introduction theory? can anyone give me an example of any other system where 2 genes like this are essential for function, may have similar function (well maybe not similar but ...
Cre Recombinase Antibody is an affinity-purified Anti-Cre rabbit antibody that can be used in Western blot or IHC detection of Cre recombinase
Cre Recombinase Antibody is an affinity-purified Anti-Cre rabbit antibody that can be used in Western blot or IHC detection of Cre recombinase
- Marengo girls basketball coach Ralph Hix could have had the best team he`s ever had entering this season. But things haven`t gone perfectly. First he lost 6-foot-3-inch sophomore center Rachel
0:07 Kinetics of Sucrose Inversion 0:30 Preparing the reactions 1:15 Polarimeter and the inversion reaction 2:45 Measuring and mixing reactants 6:30 Using polarimeter and cell 7:05 Beginning ...
0:07 Kinetics of Sucrose Inversion 0:30 Preparing the reactions 1:15 Polarimeter and the inversion reaction 2:45 Measuring and mixing reactants 6:30 Using polarimeter and cell 7:05 Beginning ...
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Chirurgische Intensivmedizin. A.D. Niederbichler; K. Ipaktchi; A. Jokuszies; T. Hirsch; M.A. Altintas; A.E. Handschin; K.H. Busch; M. Gellert; H.-U. Steinau; P.M. Vogt; L. Steinsträsser // Der Chirurg;Oct2009, Vol. 80 Issue 10, p934 Zusammenfassung  Das septische Krankheitsbild zeichnet sich durch eine enorme Dynamik aus. Dies macht sofortiges Einschreiten notwendig, um das Fortbestehen der pathologischen Systemdysregulation zu unterbinden und ein Fortschreiten der Erkrankung hin zum septischen Schock und... ...
The report generally describes terminal deoxynucleotidyl transferase, examines its uses, production methods, patents. TERMINAL DEOXYNUCLEOTIDYL TRANSFERASE
0197] This aspect of the disclosure includes the introduction of FRT sites that may be used in the FLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system. For example, see Lyznik, et al., Site-Specific Recombination for Genetic Engineering in Plants, Plant Cell Rep (2003) 21:925-932 and WO 99/25821, which are hereby incorporated by reference. Other systems that may be used include the Gin recombinase of phage Mu (Maeser et al., 1991; Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag 1994), the Pin recombinase of E. coli (Enomoto et al., 1983), and the R/RS system of the pSR1 plasmid (Araki et al., 1992). 6. Genes that affect abiotic stress resistance (including but not limited to flowering, ear and seed development, enhancement of nitrogen utilization efficiency, altered nitrogen responsiveness, drought resistance or tolerance, cold resistance or tolerance, and salt resistance or tolerance) and increased yield under stress. For example, see: WO 00/73475 where water use ...
Gamma-delta resolvase is the prototype of a large family of site-specific recombinases that use a specific serine residue as the nucleophile for cutting and rejoining defined DNA segments. The serine recombinases make concerted double strand breaks in the two recombination sites before any exchange and resealing of DNA strands occurs. Phosphodiester bond energy is conserved by formation of a covalent resolvase-DNA (phospho-serine) linkage to the 5 ends of the transiently broken DNA strands. Gamma-delta resolvase performs site-specific recombination in an elaborate synaptic complex containing 12 resolvase subunits and two 114 base pair DNA segments (called res) each with three specific dimer binding sites. We recently proposed a new model for the synaptic complex, using a combination of structural information and a detailed analysis of the various interactions between resolvase protomers that are responsible for the assembly and function of the active complex. A strong implication of the model ...
Figure 1. -Schematic diagram of mitotic recombination occurring in eye precursor cells with the ey-GAL4/UAS-FLP/GMR-hid (EGUF/hid) method. At the far left, a G2 premitotic photoreceptor precursor cell is depicted that is undergoing FLP-mediated mitotic recombination between nonidentical (homologous) chromosome arms. Premitotic photoreceptor precursors that undergo an even number of this type of recombination event follow the pathway indicated by the downward arrow and do not give rise to homozygous progeny cells. Premitotic photoreceptor precursors that undergo an odd number of these recombination events follow the pathway indicated by the upward arrow and have a 50% chance of giving rise to either heterozygous or homozygous progeny cells depending on the chromosome segregation pattern at cell division. "Silent" G2 recombination events between identical (sister) chromosome arms as well as G1 recombination events between homologous chromosomes will also be occurring, but are inconsequential with ...
Buy our Recombinant HIV1 integrase protein. Ab49075 is a protein fragment produced in Escherichia coli and has been validated in WB, ELISA. Abcam provides free…
0082]Summarized in Table 5 below are data from experiments in which target lines created using the plasmids described in Table 2 were bombarded with a substrate plasmid containing a GUS reporter gene flanked by the corresponding FRT sites used in the target constructs. This experiment measured the ability to detect transient GUS expression shortly after introduction of the substrate plasmid. Since there is no promoter in front of the first coding sequence in the substrate plasmids, random integration, unless occurring in frame behind an appropriate regulatory sequence elsewhere in the genome, would not result in GUS expression. This assay system then evaluates the ability to target FRT-flanked genes into FRT sites in the genome. In general, FRT substrate vectors (Table 6) are constructed as promoterless FRT/gene fusions cloned using the 5 flanking NheI site of the FRT to remove the ATG start codon. Genes fused in frame to the FRT with the flanking XhoI site include one of several scorable or ...
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 ...
Generation of ZnT8KO mice. Gene targeting in ES cells was designed to delete exon 5 of the endogenous Slc30a8 locus (Figure 1A). The targeting vector contained exon 5 flanked by loxP sites and an frt-flanked neocassette (Pr-Neo pA) in the 3′-adjacent region. Vector electroporation into TT2 ES cells (72), positive-negative selection, and Southern blot analysis (data not shown) yielded frt-Neor heterozygous ES cell clones. These cells were injected into CD-1 8-cell-stage embryos to generate chimeric mutant mice. The neocassette was excised in vivo by crossing the chimeras to mice expressing the Flp recombinase (B6-Tg [CAG-FLPe36]; ref. 73), leading to Slc30a8f/+ offspring (accession no. CDB0625K; http://www.cdb.riken.jp/arg/mutant%20mice%20list.html). Slc30a8f/+ mice were then backcrossed onto the C57BL6/J background more than 10 times. The resulting Slc30a8f/f mice were bred with RIP-cre transgenic mice to generate ZnT8KO mice, with β cell-specific Slc30a8 deletion. Mice were housed in a ...
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DNA Nucleotidylexotransferase: A non-template-directed DNA polymerase normally found in vertebrate thymus and bone marrow. It catalyzes the elongation of oligo- or polydeoxynucleotide chains and is widely used as a tool in the differential diagnosis of acute leukemias in man. EC 2.7.7.31.
4IQT: New nucleotide-competitive non-nucleoside inhibitors of terminal deoxynucleotidyl transferase: discovery, characterization, and crystal structure in complex with the target.