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111.
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Starting in 1991, the advance of Tyr-recombinases Flp and Cre enabled superior strategies for the predictable insertion of transgenes into compatible target sites of mammalian cells. Early approaches suffered from the reversibility of integration routes and the fact that co-introduction of prokaryotic vector parts triggered uncontrolled heterochromatization. Shortcomings of this kind were overcome when Flp-Recombinase Mediated Cassette Exchange entered the field in 1994. RMCE enables enhanced tag-and-exchange strategies by precisely replacing a genomic target cassette by a compatible donor construct. After “gene swapping” the donor cassette is safely locked in, but can nevertheless be re-mobilized in case other compatible donor cassettes are provided (“serial RMCE”). These features considerably expand the options for systematic, stepwise genome modifications. The first decade was dominated by the systematic generation of cell lines for biotechnological purposes. Based on the reproducible expression capacity of the resulting strains, a comprehensive toolbox emerged to serve a multitude of purposes, which constitute the first part of this review. The concept per se did not, however, provide access to high-producer strains able to outcompete industrial multiple-copy cell lines. This fact gave rise to systematic improvements, among these certain accumulative site-specific integration pathways. The exceptional value of RMCE emerged after its entry into the stem cell field, where it started to contribute to the generation of induced pluripotent stem (iPS-) cells and their subsequent differentiation yielding a variety of cell types for diagnostic and therapeutic purposes. This topic firmly relies on the strategies developed in the first decade and can be seen as the major ambition of the present article. In this context an unanticipated, potent property of serial Flp-RMCE setups concerns the potential to re-open loci that have served to establish the iPS status before the site underwent the obligatory silencing process. Other relevant options relate to the introduction of composite Flp-recognition target sites (“heterospecific FRT-doublets”), into the LTRs of lentiviral vectors. These “twin sites” enhance the safety of iPS re-programming and -differentiation as they enable the subsequent quantitative excision of a transgene, leaving behind a single “FRT-twin”. Such a strategy combines the established expression potential of the common retro- and lentiviral systems with options to terminate the process at will. The remaining genomic tag serves to identify and characterize the insertion site with the goal to identify genomic “safe harbors” (GOIs) for re-use. This is enabled by the capacity of “FRT-twins” to accommodate any incoming RMCE-donor cassette with a compatible design. 相似文献
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Ewelina Węglarz-Tomczak Marcin Poręba Anna Byzia Łukasz Berlicki Bogusław Nocek Rory Mulligan Andrzej Joachimiak Marcin Drąg Artur Mucha 《Biochimie》2013
Neisseria meningitides is a gram-negative diplococcus bacterium and is the main causative agent of meningitis and other meningococcal diseases. Alanine aminopeptidase from N. meningitides (NmAPN) belongs to the family of metallo-exopeptidase enzymes, which catalyze the removal of amino acids from the N-terminus of peptides and proteins, and are found among all the kingdoms of life. NmAPN is suggested to be mostly responsible for proteolysis and nutrition delivery, similar to the orthologs from other bacteria. 相似文献
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Yan hong Gao Ying YuXiao gang Hu Yu juan CaoJian zhong Wu 《Analytical biochemistry》2013,440(2):205-211
Hormones regulate the mechanism of plant growth and development, senescence, and plants’ adaptation to the environment; studies of the molecular mechanisms of plant hormone action are necessary for the understanding of these complex phenomena. However, there is no measurable signal for the hormone signal transduction process. We synthesized and applied a quantum dot-based fluorescent probe for the labeling of jasmonic acid (JA) binding sites in plants. This labeling probe was obtained by coupling mercaptoethylamine-modified CdTe quantum dots with JA using N-hydroxysuccinimide (NHS) as a coupling agent. The probe, CdTe–JA, was characterized by transmission electron microscopy, dynamic light scattering, and fluorescent spectrum and applied in labeling JA binding sites in tissue sections of mung bean seedlings and Arabidopsis thaliana root tips. Laser scanning confocal microscopy (LSCM) revealed that the probe selectively labeled JA receptor. The competition assays demonstrated that the CdTe–JA probe retained the original bioactivity of JA. An LSCM three-dimensional reconstruction experiment demonstrated excellent photostability of the probe. 相似文献
119.
The tailing genome walking strategies are simple and efficient. However, they sometimes can be restricted due to the low stringency of homo-oligomeric primers. Here we modified their conventional tailing step by adding polythymidine and polyguanine to the target single-stranded DNA (ssDNA). The tailed ssDNA was then amplified exponentially with a specific primer in the known region and a primer comprising 5′ polycytosine and 3′ polyadenosine. The successful application of this novel method for identifying integration sites mediated by φC31 integrase in goat genome indicates that the method is more suitable for genomes with high complexity and local GC content. 相似文献
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Marta Spodzieja Monika Rafalik Aneta Szymańska Aleksandra S. Kołodziejczyk Paulina Czaplewska 《Journal of molecular recognition : JMR》2013,26(9):415-425
Secondary amyloid A (AA) amyloidosis is an important complication of some chronic inflammatory diseases, primarily rheumatoid arthritis (RA). It is a serious, potentially life‐threatening disorder caused by the deposition of AA fibrils, which are derived from the circulatory, acute‐phase‐reactant, serum amyloid A protein (SAA). Recently, a specific interaction between SAA and the ubiquitous inhibitor of cysteine proteases—human cystatin C (hCC)—has been proved. Using a combination of selective proteolytic excision and high‐resolution mass spectrometry, the binding sites in the SAA and hCC sequences were assessed as SAA(86–104) and hCC(96–102), respectively. Here, we report further details concerning the hCC–SAA interaction. With the use of affinity tests and florescent ELISA‐like assays, the amino acid residues crucial for the protein interaction were determined. It was shown that all amino acid residues in the SAA sequence, essential for the formation of the protein complex, are basic ones, which suggests an electrostatic interaction character. The idea is corroborated by the fact that the most important residues in the hCC sequence are Ser‐98 and Tyr‐102; these residues are able to form hydrogen bonds via their hydroxyl groups. The molecular details of hCC–SAA complex formation might be helpful for the design of new compounds modulating the biological role of both proteins. Copyright © 2013 John Wiley & Sons, Ltd. 相似文献