Bethesda, MD 20894, Copyright Stehle IM, Postberg J, Rupprecht S, Cremer T, Jackson DA, Lipps HJ: Establishment and mitotic stability of an extra-chromosomal mammalian replicon. 10.1248/bpb.29.1294. Schaarschmidt D, Baltin J, Stehle IM, Lipps HJ, Knippers R: An episomal mammalian replicon: sequence-independent binding of the origin recognition complex. Baiker A, Maercker C, Piechaczek C, Schmidt SB, Bode J, Benham C, Lipps HJ: Mitotic stability of an episomal vector containing a human scaffold/matrix-attached region is provided by association with nuclear matrix. For rescue experiments of cell culture materials, chromosomal DNA of stably selected mixed clones was isolated three times independently and transformed into bacteria. 10.1038/sj.mt.6300399. an EGFP-BSD cassette connected via a recently described, 22 bp long internal ribosomal entry site (EGFP-IRES-BSD) [25]. Increased and prolonged expression of pEPito in vivo. For the latter cell line rapid silencing of exogenous plasmid DNA by histone deacetylation has been frequently observed [42, 43]. 2000, 403: 795-800. 10.1038/321209a0. When selecting HEK293 cells transfected with the eight different vector constructs, stably selected mixed-clone cells could be obtained in all cases (Figure 4A, C). It was shown, that the CpG content of a minicircles' transgene transcription unit does not affect the epigenetic silencing of its transgene [37], in contrast to epigenetic elements present in the bacterial origin of replication or backbone [38]. Nat Biotechnol. As described above for the pEPI-1 and pEPI-1-ΔMARS vector backbones, the individual functional vector elements could be inserted into pHulk by restriction digest and ligation using PciI/NheI (promoter elements), NheI/BglII (transgene transcription units), or BglII/MluI (MARS). Standard error of the mean for each time point is indicated. Expression levels and colony formation efficiencies in stably selected HEK293 and NIH3T3 cells. Eisenberger T, Lipps HJ, Mueller M, Baiker A: A human papillomavirus (HPV)-based pseudoviral gene delivery system for the non-viral, episomally replicating vector pEPI-1. Vectors based on the pEPito-ΔMARS or pEPI-1 vector backbones exhibited a ~10 fold increased colony-forming efficiency as compared to the vectors based on the pEPI-1-ΔMARS vector backbones in HEK293 cells, indicating a positive effect of the CpG depleted pEPito-ΔMARS vector backbone or the presence of a MARS element on colony-forming efficiency (Figure 4C). After sacrificing all animals at 32 d.p.i., chromosomal DNA was extracted from their livers. Un estudio comparativo sobre el español y el sueco [Spatio-temporal metaphors and time perception: a … In conclusion, we developed a CpG-depleted pEPI-vector derivative named pEPito in this study which combines the ability of episomal replication and mitotic stability described for pEPI-based vectors with the diminished tendency towards epigenetic silencing of vector encoded transgenes due to the CpG depletion of the vector backbone. The majority of human promoters belong to the high-CpG class of promoters, for which it is well established that the methylation status of the surrounding CpG islands has a direct influence on the promoter activity unmethylated CpG islands in the promoter region correspond to active promoters, whereas heavily methylated mCpG islands correspond to inactive promoters [18]. On HEK293 cells, all pEPito-based vectors exhibited transfection efficiencies of around 75%, independent of plasmid size, promoter type, or the presence or absence of the MARS element, whereas with all pEPI-1 based vectors 45-65% EGFP positive cells were found (Figure 3A). The episomal status of all four MARS bearing vectors could be verified by bacterial rescue experiments using chromosomal DNA extracted from stably selected HEK293 mixed-clones. Episomal minicircles persist in periods of transcriptional inactivity and can be transmitted through somatic cell nuclear transfer into bovine embryos. Mol Biol Rep. 2004, 31: 85-90. 2002, 5: 731-738. Antivirus McAfee Total Protection 2021 – 5 Appareils – 1 an. Vectors based on the pEPI-1 backbones contain a pUC Ori for bacterial propagation, a MARS, a second mammalian SV40-O/P driven NPT transcription unit for bacterial kanamycin or mammalian geneticin (G418) selection purposes, and a total of 206 CpG motives. Taken together, these findings suggest that a state of the art plasmid vector for gene delivery into mammalian cells should be composed of a promoter element that is depleted of CpG islands in order to minimize silencing phenomena, and a CpG depleted vector backbone to prevent undesired stimulatory effects on the innate immune system [16, 22]. 2008, 16: 548-556. The resulting shuttle vector (pHulk) served as a template for the construction of all pEPito and pEPito-ΔMARS vectors. pEPito- [CMV-IEP]- [EGFP::Luc] and pEPito- [hCMV/EF1P]- [EGFP::Luc], could be rescued from chromosomal liver DNA preparations without any rearrangements at 32 d.p.i., indicating that the presence of a MARS element within the pEPito vector backbone facilitates the persistence of episomes in a "rescue"-able form in vivo. The vector pEPI-1- [CMV-IEP]- [Luc] (Figure 1E, #9) has been described earlier [32]. background, integration of few pEPI-vectors was never fully excluded, but the episomal maintenance and replication was described [1–4, 6–9]. Dual-Acting Antiangiogenic Gene Therapy Reduces Inflammation and Regresses Neovascularization in Diabetic Mouse Retina. Chen et al. de Wolf HK, Johansson N, Thong AT, Snel CJ, Mastrobattista E, Hennink WE, Storm G: Plasmid CpG depletion improves degree and duration of tumor gene expression after intravenous administration of polyplexes. The resulting first shuttle vector (pHulk-ΔMCS) consisted of a CpG free R6Kori, a CpG depleted BLA cassette, and a XhoI single cutter site. Springer Nature. Hum Mol Genet. 2004, 1: 115-125. Results: 2. 2004, 23: 191-201. Finito pepito! And on an even perkier day, precisely on the 8th of September 2016, Judge (Pepito) Rodriguez, Mr. Danner, and I met at the Citrus Club in downtown Orlando. EMBO Rep. 2002, 3: 349-354. 10.1074/jbc.M303495200. Bode J, Benham C, Knopp A, Mielke C: Transcriptional augmentation: modulation of gene expression by scaffold/matrix-attached regions (S/MAR elements). 2006, 10: 233-244. Nat Genet. Mol Cell Biol. In these studies partial, i.e. 1999;27:426–428. Furthermore, pEPI-1 consists of two mammalian transcription units oriented in a clockwise direction. Furthermore we like to thank Helga Mairhofer and Georg Malterer for their help with real time PCR and Anja Ehrhardt for her help with Southern blotting. The expression profile of CMV-IEP, however, decreases steadily over time, which could be explained by methylation of cytosines within this promoter region [19]. Luciferase activity was quantified after 1, 7, 14, 21 (data not shown) and 32 days post injection (d.p.i.) statement and Methylation of CpG islands within the CMV-IEP region has been suggested as one explanation for this long-known, but undesired silencing phenomenon [19], even if recent publications indicate other mechanisms for this effect [20, 21]. 10.1126/science.1553545. Additionally, the performance of all vectors is improved when replacing the CMV-IEP by the human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element, that is known to be less affected by epigenetic silencing events [17]. Conclusions: In this study we present an episomal plasmid system designed for tissue specific transgene expression and replication. Here, we report the development of a novel vector pEPito, which is derived from the pEPI-1 plasmid replicon but has considerably improved efficacy both in vitro and in vivo. Mol Ther. 10.1517/17425247.1.1.115. Luciferase expression is quantified using Xenogen Living Image software and represented as photons/sec/cm2/sr. Google Scholar. Genome-wide profiling of S/MAR-based replicon contact sites. Rude or colloquial translations are usually marked in red or orange. doi: 10.1093/nar/27.2.426. On a écrit une histoire, contacté Zaffiro, et après s’être mis d’accord avec lui sur le style, les univers et les expressions, on s’est lancés dans une collaboration riche en rebondissements. [40]. 2004, 6: 395-404. 2002, 32: 370-377. The Xenogen system reported bioluminescence as photons/sec/cm2/sr (seradian) in a 2.86-cm-diameter region of interest covering the liver. All novel vectors comprised an identical transgene, i.e. Nehlsen K, Broll S, Bode J: Replicating minicircles: Generation of nonviral episomes fort the efficient modification of dividing cells. BMC Biotechnol 10, 20 (2010). Las metáforas espacio-temporales y la percepción del tiempo. National Library of Medicine Transformed bacteria were selected on LB-plates containing either ampicillin, or kanamycin depending on respective bacterial selection markers (Table 1). Imai S, Armstrong CM, Kaeberlein M, Guarente L: Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. A multiple cloning site consisting of the restriction enzyme sites XhoI, PciI, NheI, BglII, MluI, and XhoI was generated by primer annealing and inserted into the single XhoI site of pHulk-ΔMCS. 2006, 29: 1294-1296. Grey bars indicate vectors containing an hCMV/EF1P promoter element, black bars indicate vectors containing a CMV-IEP promoter element. Since liver cells are quiescent cells, episomal replication of pEPI-based vectors might not serve as a suitable explanation for the prolonged transgene expression. This is noteworthy, since all previous pEPI-1 based vectors were selected for the NPT gene, present in the second dispensable SV40-O/P driven transcription unit [1, 3, 4]. Luciferase expression profiles of exemplary MF-1 mice hydrodynamically injected with the vectors #9-13 (Figure 2 as assayed by in vivo bioluminescence imaging after 1 (first column), 7 (second column), 14 (third column), and 32 (fourth column) days post injection. The known establishment efficiency for pEPI-1-replicons is about 1-5% [8]. -, Jenke AC, Stehle IM, Herrmann F, Eisenberger T, Baiker A, Bode J, Fackelmayer FO, Lipps HJ. COVID-19 is an emerging, rapidly evolving situation. The plasmids pEPito- [hCMV/EF1P]- [EGFP-IRES-BSD] and pEPito- [CMV-IEP]- [EGFP-IRES-BSD] could be rescued from chromosomal DNA extracted from stably selected NIH3T3 cells, but no MARS depleted control vector could be rescued (Table 1). 10.1093/nar/27.2.426. This figure does not claim proportional correctness. ... you held up Finito … Privacy 2003, 278: 33793-33800. The pEPito backbones contain a R6K Ori [24] for bacterial propagation, a β-lactamase gene (BLA) for bacterial ampicillin selection, a MARS, no second mammalian transcription unit, and only 37 CpG motives. 2008, 26: 549-551. speculate that there might be a recruitment of repressive heterochromatin as inducer of a silencing complex that is initiated within bacterial vector elements [20], implying an important role of the chromatin structure for epigenetic silencing. Idilio Trunco . 10.1002/jgm.516. It is what it is—the physical expression of Golden Age music shared within the crowded, social confines of a milonga. 2017 Jul 27;45(13):7841-7854. doi: 10.1093/nar/gkx522. Mol Hum Reprod. In this manuscript we present a novel pEPI-vector derivative, named pEPito, with higher transgene expression levels and increased colony-forming efficiencies in vitro, and more persistent transgene expression profiles in vivo. The differences in transient EGFP expression, the EGFP expression levels of stably selected mixed-clones, and the 7-70 fold increase in colony-forming efficiency are impressive and not caused by the different equimolar levels of the transfected DNA, which differ at a maximum of 33%. The human CMV enhancer/human elongation factor 1 alpha promoter (hCMV/EF1P) element was PCR-amplified out of pCPG-hCMV-Luc using 5' NheI and 3' BglII primers. As indicated in Table 1, the two pEPito based vectors with MARS element, i.e. 2008 Oct;8(5):353-66. doi: 10.2174/156652308786071032. Additional file 1: figure 3A. (A, B) Mean EGFP expression levels per cell (A: HEK293, B: NIH3T3) of stably selected cells. Boyes J, Bird A: Repression of genes by DNA methylation depends on CpG density and promoter strength: evidence for involvement of a methyl-CpG binding protein. Wang XY, Zhang X, Wang TY, Jia YL, Xu DH, Yi DD. Anal Biochem. pEPito: a significantly improved non-viral episomal expression vector for mammalian cells. Conclusions: In eukaryotic DNA, CpG dinucleotides in eukaryotes appear at lower frequency and are often methylated to 5-methyl-cytosine (mCpG), whereas in most bacterial genomes, however, CpG dinucleotides are represented according to their statistical expectation, and the cytosine residue is normally unmethylated [13, 14]. Dan Boccia here from Anchorage. Ochiai H, Harashima H, Kamiya H: Silencing of exogenous DNA in cultured cells. 10.1038/35004061. the formation of heterochromatin after histone deacetylation involvement [44–47] triggered by certain bacterial elements, may serve as an explanation for the better performance of pEPito based vectors with respect to transgene expression and colony-forming efficiency within stably selected NIH3T3 cells (Figure 4B, D), since the pEPI-1 vector backbones exhibit significantly higher numbers of CpG motives (Figure 2). All 13 different vectors used within this work were constructed, propagated, and amplified in E.coli DB3.1λpir [30]. (B) Nuclear retention and mitotic stability of pEPI-based vectors can be explained by MARS mediated direct or indirect interaction with metaphase chromosomes in a "piggy back"-like mechanism. 10.1083/jcb.200303047. The detailed molecular mechanism of the epigenetic silencing of vector encoded transgenes has to be further investigated. Nature. 1,190 Followers, 293 Following, 11 Posts - See Instagram photos and videos from abdou now online (@abdoualittlebit) Generation of a tumor- and tissue-specific episomal non-viral vector system. 1992, 255: 195-197. In case of HEK293 cells the results of the colony-forming assays performed are presented as fold increase in relation to pEPI-1- [CMV-IEP]- [EGFP-IRES-BSD]-ΔMARS (Figure 4C). The Cytomegalovirus immediate early promoter (CMV-IEP) element was isolated as PciI and NheI fragment out of pEGFP-C1 (Clontech, USA). [20], i.e. Pepito Avellanada (Pepito de Avellaneda) : grand danseur de milonga, originaire du quartier de Avellaneda. The latter (BLA) cassette was PCR-amplified from pMOD-LucSH in a way that PacI restriction sites were added to either ends by primer mutagenesis. Pringle IA, Raman S, Sharp WW, Cheng SH, Hyde SC, Gill DR: Detection of plasmid DNA vectors following gene transfer to the murine airways. Navegue no maior catálogo de séries da internet e acompanhe as suas favoritas!. Jenke BH, Fetzer CP, Stehle IM, Jonsson F, Fackelmayer FO, Conradt H, Bode J, Lipps HJ: An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo. 2020 Feb;38(2):151-157. doi: 10.1038/s41587-019-0402-x. Stehle IM, Scinteie MF, Baiker A, Jenke AC, Lipps HJ: Exploiting a minimal system to study the epigenetic control of DNA replication: the interplay between transcription and replication. Cite this article. See this image and copyright information in PMC. Explorations in the left periphery: The expression of polarity emphasis ... Pepito bien ha comido pasta. All vectors contain either the CMV-IEP or the hCMV/EF1P promoter element. All animals exhibited an equally strong luciferase signal on day one after injection, at later time points the luciferase signal steadily decreased. Epub 2019 Jan 29. In NIH3T3 cells, only vectors based on the hCMV/EF1 promoter element resulted in significant numbers of formed colonies (Figure 4D): pEPito- [hCMV/EF1P]- [EGFP-IRES-BSD] exhibited a ~100 fold increased colony-forming efficiency as compared to pEPI-1- [hCMV/EF1P]- [EGFP-IRES-BSD]-ΔMARS indicating a positive effect of the pEPito vector backbone. 2002, 420: 198-202. Gene Ther. The rather weak performance of the CMV-IEP promoter element within NIH3T3 cells has been already described by Nehlsen et al. A cytosine monophosphate (C) followed by a guanine monophosphate (G) in a nucleotide sequence is referred to as CpG dinucleotide (or CpG islands). En anglès tenim les expressions Piece of cake (literalment, tros de pastís), ... “s’ha acabat la festa”), o “E mo abbiamo proprio finito di campa” (literalment, “hem fet el camp”) per a transmetre la idea de que ja s’ha arribat al final d’alguna cosa bona, de que ja no en queda més. In case of stably selected HEK293 cells, CMV-IEP promoter based vectors exhibited a stronger transgene expression - as a measure of promoter strength - as vectors based on the relatively weaker hCMV/EF1P promoter element (Figure 4A). Our novel pEPito vector will have crucial implementations for biotechnological applications in vitro, e.g. https://doi.org/10.1186/1472-6750-10-20, DOI: https://doi.org/10.1186/1472-6750-10-20. In case no colonies could be obtained from the initial transformations, this procedure was repeated for two more times. Nat Biotechnol. 10.1023/A:1024962308071. Shortened nuclear matrix attachment regions are sufficient for replication and maintenance of episomes in mammalian cells. Par exemple: « Je n’ai jamais eu de chance dans la vie » pourra devenir “Je suis une personne chanceuse qui profite de chaque instant de la vie.” [41]. Ça fait trop longtemps qu'on Nat Cell Biol. Gene Ther Mol Biol. The first, Cytomegalovirus immediate early promoter (CMV-IEP) driven enhanced green fluorescent protein (EGFP) transcription unit is oriented into the MARS and has been shown to be a functional component of the pEPI-1 vector plasmid replicon. -, Baiker A, Maercker C, Piechaczek C, Schmidt SB, Bode J, Benham C, Lipps HJ. Prevention and treatment information (HHS). Description: This additional file depicts representative flow cytometry profiles corresponding to all bars shown in figure 4A (stably selected mixed-clone HEK293 cells). BMC Biotechnology 2005, 12: 1206-1214. Background: J Bacteriol. The integrity of vectors was verified by sequencing (AGOWA, Germany). For further analysis, stably selected mixed-clone cells were generated from initially transfected cells as batch by selection with blasticidin for 30 days. These findings suggest, that the CMV-IEP promoter element seems to be extraordinarily susceptible for epigenetic silencing within NIH3T3 cells. HJL and MO contributed in vector design and construction. It exhibits major advantages compared to the original pEPI-1 plasmid, including higher transgene expression levels and increased colony-forming efficiencies in vitro, as well as more persistent transgene expression profiles in vivo. Nat Genet. The mitotic stability of pEPI-based vectors can be explained by the MARS mediated stable interaction with metaphase chromosomes [3] (Figure 1B). 10.1038/ng1017. Nowadays he’s the major Argentinian tango representative in the world. Emerging patent landscape for non-viral vectors used for gene therapy. Proc Natl Acad Sci USA. In contrast, the two pEPito based vectors pEPito- [CMV-IEP]- [EGFP::Luc] and pEPito- [hCMV/EF1P]- [EGFP::Luc] exhibit a longer lasting luciferase expression until 32 d.p.i Obviously, the pEPito vector with the hCMV/EF1P promoter element exhibited the strongest in vivo luciferase expression 32 d.p.i., about 5 fold higher compared to the CMV-IEP in pEPito and more than a 2 log difference to pEPI-Luc indicating a positive effect of the hCMV/EF1P promoter element on prolonged transgene expression in MF-1 mice in vivo. Heureusement, ce point est amélioré dans la zone DLC, de même que les angles de vue lors des dialogues, qui sont beaucoup plus variés (autre que du champ, contre champ). Les Éditions Uruk. Akira S, Hemmi H: Recognition of pathogen-associated molecular patterns by TLR family. This strong activity is due to the presence of several transcription factor binding sites within its sequence. Bauer S, Kirschning CJ, Hacker H, Redecke V, Hausmann S, Akira S, Wagner H, Lipford GB: Human TLR9 confers responsiveness to bacterial DNA via species-specific CpG motif recognition. All authors read and approved the final manuscript. Traduction de « Fabulosa » par Los Machucambos , espagnol → français Akira S, Takeda K, Kaisho T: Toll-like receptors: critical proteins linking innate and acquired immunity. JOUR J -- FINITO PEPITO ***** Demain midi sera la date limite pour remettre vos textes. All 13 different vectors have been constructed, propagated, and amplified in E.coli DB3.1ëpir [30]. The vector replicates once per cell cycle during early S-phase, with the origin recognition complex (ORC) being able to assemble at various regions on the vector DNA [6]. 10.1007/s11095-008-9558-7. 10.1038/sj.gt.3302231. By using this website, you agree to our A post-ANOVA multiple comparison procedure (Tukey's HSD) was further performed to assess pairwise differences on expression confirmed by ANOVA with a significance level p = 0.05. Hum Gene Ther. 2013 Jun 4;13:49. doi: 10.1186/1472-6750-13-49. Immunol Lett. In both, HEK293 and NIH3T3 cells, the colony-forming efficiency of vectors with a functional pEPI-plasmid replicon, i.e. 10.1016/S1525-0016(03)00168-0. BMC Cell Biol. In NIH3T3 cells, pEPito based vectors with hCMV/EF1P promoter resulted in slightly higher EGFP expression levels as compared to the CMV-IEP (Figure 3D). The individual functional elements of the vector can be exchanged by restriction digest and ligation using PciI/NheI (promoter element), NheI/BglII (transgene transcription unit), or BglII/MluI (MARS). Data were analysed by using LIVINGIMAGE 2.50 software (Xenogen, USA). Background level of light emission on non-treated animals is 1 × 106 photons/sec/cm2/sr. For colony forming assays, transfected cells were split from 6 well plates into 75 cm2 flasks at 48 hours post transfection. The resulting PCR-fragment containing IRES-BSD was inserted via BglII and BamHI into the single BglII site of pEGFP-C1 (Clontech, USA) or pEPI-1 [1]. 10.1038/sj.emboj.7600029. Dictionary of Spanish Slang and Colloquial Expressions . The pEPito-ΔMARS vectors, the smallest vectors used, exhibited the highest transient transfection efficiency indicating either an inverted effect of the plasmid size, or an effect of the initial molar amount of DNA used for transfection or both. With the pEPI-1 based control vector pEPI-1- [CMV-IEP]- [Luc], and in the two MARS depleted pEPito control vectors pEPito- [CMV-IEP]- [EGFP::Luc]-ΔMARS and pEPito- [hCMV/EF1P]- [EGFP::Luc]-ΔMARS the luciferase signal is weakly detectable until 14 d.p.i., but is below detection limit at 32 d.p.i This result indicated a positive effect of the pEPito vector backbone and the MARS element on prolonged luciferase expression. Chen ZY, He CY, Kay MA: Improved production and purification of minicircle DNA vector free of plasmid bacterial sequences and capable of persistent transgene expression in vivo. eCollection 2020 Dec 4. The non-viral plasmid vector pEPI-1 was constructed in 1999 by Piechaczek et al [1], by cloning a 2000 bp long matrix attachment region sequence (MARS), that was derived of the human β-interferon gene cluster [2], into the commercial vector pGFP-C1 (Clontech, USA). L’accent cette année sera mis sur la danse. We could further demonstrate, that the CpG content and/or the bacterial vector backbone of any plasmid vector exhibits a strong influence on long-term transgene expression. The auto function was used to define the minimum for the scale at each time point. 2000, 74: 2671-2678. This is also the case when pEPI-1 plasmids are isolated from already established mammalian cell clones and reintroduced into cells demonstrating that the primary sequence of DNA is not involved in the establishment process [8, 11, 12] but rather epigenetic features such as chromatin structure and nuclear localization [8].
The Last Shadow Puppets Lyrics, Qui Est La Mère De Lola Périer, Télécharger Podcast After Foot, Secret Warzone, Saison 5, Anne Bourdin Louis Bourdin, Zeop Rmc Sport, Cuvée Nitro Cold Brew, Coco Resumen En Español, Organiseur Mémoniak 2021, Historique Taux Livret Jeune Société Générale, Peluche Coco Disney Pixar,