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The Stress Response and Stress Proteins
Published in John J. Lemasters, Constance Oliver, Cell Biology of Trauma, 2020
Martin E. Feder, Dawn A. Parsell, Susan L. Lindquist
Can tolerance be engineered in multicellular animal models? Gene targeting is one technique that may soon provide answers to this question, but it is presently limited to a few model systems. Without targeting, however, insertion of transgenes in germ-line transformation experiments may inadvertently disrupt essential genes or alter their expression, which can obfuscate the phenotype of the transgene. Golic and Lindquist35 developed an approach that can alleviate this difficulty: The genome is transformed with a construct bearing a transgene of interest between two site-specific recombination targets from yeast. A separate transgene construct bears the region coding for the yeast FLP recombinase protein under control of an inducible promoter. When induced, FLP recombinase will catalyze recombination between sister chromatids bearing FLP recombination targets, producing a chromosome carrying multiple copies of the transgene or a chromosome lacking extra copies but interrupted at the exact same point as the extra copy chromosome. Multiple recombination events can be used to create an allelic series of chromosomes with a common point of transgene disruption but differing in transgene number. Subsequent genetic crosses can then isolate these chromosomes.
Immunoglobulins
Published in Constantin A. Bona, Francisco A. Bonilla, Textbook of Immunology, 2019
Constantin A. Bona, Francisco A. Bonilla
Many of the molecules participating in the rearrangement process have yet to be identified. Two candidates for components of a “recombinase complex” are the products of the recombinase activation genes: RAG-1 and RAG-2. Recombinase activity is an integral part of the regulation of B cell development. RAG products are prominent in young lymphocytes which are actively rearranging their Ig genes. RAG activity disappears in mature lymphocytes. Fibroblasts do not normally rearrange Ig gene segments. However, when RAG genes are introduced into these cells, they acquire the capacity for Ig gene rearrangement. It is not clear whether these genes encode recombinase components with enzymatic and/or DNA-binding activity, or if they are transcriptional regulators of genes encoding the actual recombinases.
Practical Approach to Molecular Biology in Hematopathology
Published in Harold R. Schumacher, William A. Rock, Sanford A. Stass, Handbook of Hematologic Pathology, 2019
Anwar Mikhael, Harold R. Schumacher
Assembly of both heavy- and light-chain genes involves similar molecular mechanisms. Present at the boundaries of all germline segments are consensus sequences consisting of a conserved palindromic heptamer and nanomer separated by either 12 or 23 bp of non-conserved spacer sequences. Recombination is catalysed by the recombinase (RAG-1 and RAG-2) system. In addition, terminal deoxynucleotidyl transferase (TdT) will add a few nucleotides at the junction, thereby inducing more Ig diversity (1,2).
Effects of the COVID-19 pandemic: new approaches for accelerated delivery of gene to first-in-human CMC data for recombinant proteins
Published in mAbs, 2023
Hervé Broly, Jonathan Souquet, Alain Beck
Another factor affecting cell line generation timelines is the mode of insertion of the gene(s) of interest into the host cells. Historically, transfection with a non-homologous recombination-based random integration (RI) was the common method for inserting foreign genes into a mammalian cell host to produce recombinant proteins.65 Site-specific recombinase-mediated integration (SSI) is another method.66–68 SSI has the advantage over random integration to reduce the selection and single-cell cloning stages from several weeks to a few days through negative selection (e.g., by supplementing cell culture medium with ganciclovir) to eliminate cells with unwanted recombination events, thus leading to the generation of more consistent and reliable clones.69
An insight into clinical and laboratory detections for screening and diagnosis of cervical cancer
Published in Expert Review of Molecular Diagnostics, 2023
Shruthi Padavu, Pooja Aichpure, Ballamoole Krishna Kumar, Anoop Kumar, RadhaKanta Ratho, Shipra Sonkusare, Indrani Karunasagar, Iddya Karunasagar, Praveen Rai
The recombinase polymerase amplification (RPA) has evolved as a novel isothermal amplification technique for the molecular diagnosis of HR-HPV. The RPA, unlike many other isothermal methods, does not require high temperatures and proceeds in the range of 37–42°C within 20–30 minutes [75]. This technique employs the recombinase activity and strand-displacing activity of the enzyme to unwind dsDNA and amplify the target DNA, respectively. The RPA technique is quite helpful in detecting HR-HPV because of its rapidity and near-normal reaction temperature. The end-point of the isothermal amplification product can be interpreted using fluorescent-based nucleic acid detection or gel electrophoresis. End-point detection can also be achieved by chemically labeling the RPA reaction and using lateral flow strips that enable instrument-free signal readouts. TwistAmp® (TwistDx™, UK) offers a variety of RPA kits with varying end-point detection for research and commercial applications [76]. The RPA achieves exponential amplification without the necessity for the pretreatment of the DNA sample. The RPA assay was shown to be a powerful approach for detecting HPV and may be a beneficial tool for the early diagnosis of HPV infection in prognostic applications [77].
An overview of human leptospirosis vaccine design and future perspectives
Published in Expert Opinion on Drug Discovery, 2020
Carolina R. Felix, Bianca S. Siedler, Liana N. Barbosa, Gabriana R. Timm, Johnjoe McFadden, Alan J. A. McBride
Surprisingly, cytosolic proteins have also been shown to elicit protective immunity even though they are not the logical choice for vaccine candidates as they are not generally thought to be expressed on the bacterial surface. Recently, recombinase A (RecA), a cytosolic protein, protected 83% of immunized animals against homologous and heterologous challenges and induced sterilizing immunity [44]. Although several antigens have elicited sterilizing immunity (Table 1), this is not generally considered a priority for a leptospiral vaccine since person-to-person transmission of leptospirosis is not considered to be a risk [27]. As the search for novel protective candidates is limited by several logistical constraints, including the indispensable use of experimental animals and funding, the field has tended to focus on OMPs, as these are more likely to elicit protective immunity than cytosolic proteins.