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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.
Use of Enzymes in the Downstream Processing of Biopharmaceuticals
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Precise enzyme-catalyzed structural modifications have also targeted key impurities with the goal of facilitating their separation from the target molecule. Two notable examples can be found in the case of the very challenging purification of minicircle (MC) DNA vectors. MCs are produced in vivo in Escherichia coli by first replicating a parental plasmid (PP) backbone and then promoting an intramolecular site-specific recombination between two strategically placed multimer resolution sites (Simcikova et al., 2016). This generates a replication-deficient MC, which contains the expression cassette with therapeutically useful transgene, and a miniplasmid (MP), which contains the prokaryotic replication segment used for PP amplification. The separation of MCs from the MP impurities is very difficult to perform since the two species are very similar in terms of structure and size (Alves et al., 2016).
Recombinant Antibodies
Published in Siegfried Matzku, Rolf A. Stahel, Antibodies in Diagnosis and Therapy, 2019
Melvyn Little, Sergey M. Kipriyanov
Recombinant antibody technology is opening up new perspectives for the development of novel therapeutic and diagnostic agents. For example, human antigen binding fragments have been derived from lymphocyte gene repertoires and from libraries of synthetic antibodies. To screen such libraries containing many millions of different clones, a selection system is required with an efficiency comparable to that of the immune system. This has been achieved by displaying antibodies on the surface of phage particles containing the antibody’s gene, analogous to the expression of the IgM antigen receptor on the surface of inactivated B-lymphocytes. Millions of different clones from complex libraries can be panned simultaneously over an immobilized substrate followed by amplification of the adherent phages. This process might be made even more efficient in future by exploiting the antibody-mediated infection of E. coli, an evolutionary principle similar to that governing clonal selection in the immune system. The size of the antibody libraries displayed on phage particles has, until recently, been restricted by the efficiency of in vitro packaging in E. coli. Using the Cre-lox site-specific recombination system described above (Waterhouse et al., 1993; Griffiths et al., 1994), a phage display library could theoretically be generated containing as many different antibodies as the number of E. coli in culture.
Flagellum and toxin phase variation impacts intestinal colonization and disease development in a mouse model of Clostridioides difficile infection
Published in Gut Microbes, 2022
Dominika Trzilova, Mercedes A. H. Warren, Nicole C. Gadda, Caitlin L. Williams, Rita Tamayo
Many bacterial species employ phase variation to generate phenotypic heterogeneity within a clonal population. Bacteria frequently encounter selective pressures in their environment, and phenotypic heterogeneity helps ensure survival by creating subpopulations that are differentially equipped to overcome these pressures.1 Phase variation typically affects the production of surface factors that directly interface with the bacterium’s environment, such as flagella, pili, and exopolysaccharides. Both mucosal pathogens and commensal species employ phase variation to balance the fitness advantages conferred by these structures with the costs of producing them; in a host environment, the ability to phase vary can promote immune evasion and persistence in the host.2 Phase variation can be achieved by multiple epigenetic and genetic mechanisms, including DNA modification by methylation, slipped-strand mispairing, homologous recombination, and site-specific recombination.1,3
Long-peptide vaccination with driver gene mutations in p53 and Kras induces cancer mutation-specific effector as well as regulatory T cell responses
Published in OncoImmunology, 2018
Jasmin Quandt, Christoph Schlude, Michael Bartoschek, Rainer Will, Angel Cid-Arregui, Sebastian Schölch, Christoph Reissfelder, Jürgen Weitz, Martin Schneider, Stefan Wiemann, Frank Momburg, Philipp Beckhove
For the generation of 2277-NS mutant Tp53/Kras transgene expressing sublines, FRT expression vectors containing a hygromycin (InvitrogenTM, Life technologies, 10687–010) resistance gene, tet repressor gene (tetR) as well as a tetracycline-responsive CMV promoter were cloned upstream of the wt and mutant chimeric mutant Tp53/Kras transgenes’ ORFs, respectively, for inducible transgene expression. After sequence verification, the wt and mutated constructs were individually co-transfected together with a Flp recombinase expression vector (pOG44, InvitrogenTM, Life technologies, V6005-20) to integrate the respective expressions constructs into the mouse Rosa26 locus of the 2277-NS acceptor cell line by site specific recombination. After selection (hygromycin positive/eGFP negative), single colonies were picked and analyzed.
Identification of a Novel 9.7 kb Deletion Causing α0-Thalassemia in Two Pregnant Women in Southern China
Published in Hemoglobin, 2018
Li Lin, Qi Yang, Shujie Zhang, Yangjng Zuo, Sheng He, Xiangmin Xu, Biyan Chen, Xiaoxia Qiu
The novel rearrangement in the α-globin gene cluster that likely arose due to a similar nonallelic homologous recombination (NAHR) [9,10]. According to the sequencing result, the 5′ deletion sequence near to the breakpoint was the same 6 bp as the 3′ sequence near to the breakpoint. This novel deletion might be generated by a site-specific recombination [3]. The hematological results of the two heterozygous probands indicated that the MCV and MCH levels of the deletion were both decreased when compared with normal people and close to the level of a – –SEA/αα carrier. Although the proband from Family B had mild anemia with a normal ferritin level, it probably resulted from hypervolemia due to pregnancy. All evidence shows that the – –del9710/αα is an α0-thal mutation.