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Journal articles on the topic 'EGFP-TAG'

Author: Grafiati

Published: 4 June 2021

Last updated: 25 May 2024

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1

Tandio Saputro, Shania Safera, Khayu Wahyunita, Astutiati Nurhasanah, et al. "Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1." F1000Research 12 (March 27, 2024): 1. http://dx.doi.org/10.12688/f1000research.123181.2.

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Background The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods The study aimed to investigate the optimiz

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2

Tandio Saputro, Shania Safera, Khayu Wahyunita, Astutiati Nurhasanah, et al. "Expression of modified enhanced green fluorescent polyarginine protein in Saccharomyces cerevisiae INVSc1." F1000Research 12 (January 3, 2023): 1. http://dx.doi.org/10.12688/f1000research.123181.1.

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Background: The enhanced green fluorescent protein (EGFP) gene is a reporter gene that can be used to optimize protein isolation procedures and the functional working of a transduction protein. EGFP, with the addition of eleven arginine residues, has been engineered to functionally improve the protein transduction process, which can later be used for cell reprogramming like induced pluripotent stem cells. The addition of six histidine amino acid residues at its C-terminal is intended for the protein isolation process using the His-tag antibody. Methods: The study aimed to investigate the optim

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3

Hänisch, Jan, Marc Wältermann, Horst Robenek, and Alexander Steinbüchel. "The Ralstonia eutropha H16 phasin PhaP1 is targeted to intracellular triacylglycerol inclusions in Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, and provides an anchor to target other proteins." Microbiology 152, no. 11 (2006): 3271–80. http://dx.doi.org/10.1099/mic.0.28969-0.

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In Ralstonia eutropha, the H16 phasin PhaP1 represents the major phasin that binds to the surface of polyhydroxyalkanoate (PHA) inclusions. In this study, C-terminal fusions of PhaP1 with enhanced green fluorescent protein (eGFP) and with Escherichia coli β-galactosidase (LacZ) were expressed separately in the triacylglycerol (TAG)-accumulating actinomycetes Rhodococcus opacus PD630 and Mycobacterium smegmatis mc2155, employing the M. smegmatis acetamidase (ace) promoter of the Escherichia–Mycobacterium/Rhodococcus shuttle plasmid pJAM2. PhaP1 and the PhaP1 fusion proteins were expressed stabl

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4

Klimecka, Maria Magdalena, Anna Antosiewicz, Matylda Anna Izert, et al. "A Uniform Benchmark for Testing SsrA-Derived Degrons in the Escherichia coli ClpXP Degradation Pathway." Molecules 26, no. 19 (2021): 5936. http://dx.doi.org/10.3390/molecules26195936.

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The ssrA degron is commonly used in fusion proteins to control protein stability in bacteria or as an interaction module. These applications often rely on the modular activities of the ssrA tag in binding to the SspB adaptor and in engaging the ClpXP protease. However, a comparison of these activities for a substantial standard set of degron variants has not been conducted previously, which may hinder the development of new variants optimized exclusively for one application. Here, we strive to establish a benchmark that will facilitate the comparison of ssrA variants under uniform conditions.

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Lu, Wei, Ruolin Wang, Pan Wang, Sanyuan Ma, and Qingyou Xia. "Genetic Code Expansion System for Tight Control of Gene Expression in Bombyx mori Cell Lines." Insects 12, no. 12 (2021): 1081. http://dx.doi.org/10.3390/insects12121081.

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Inducible gene expression systems are important tools for studying gene function and to control protein synthesis. With the completion of the detailed map of the silkworm (Bombyx mori) genome, the study of Bombyx mori has entered the post-genome era. While the functions of many genes have been described in detail, many coding genes remain unidentified. Except for the available tetracycline induction system, there is currently a dearth of other effective induction systems for B. mori. A genetic code expansion system can be used for protein labeling and to regulate gene expression. Here, we have

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Kwon, Sojung, Areum Kwak, Hyejin Shin, Soyoung Choi, Soohyun Kim, and Hyunjung Jade Lim. "Application of a novel cell-permeable peptide-driven protein delivery in mouse blastocysts." REPRODUCTION 146, no. 2 (2013): 145–53. http://dx.doi.org/10.1530/rep-13-0203.

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Cell-permeable peptides (CPPs) mediate the delivery of macromolecules into cells. However, whether CPPs are usable in mammalian oocytes and embryos for the modulation of protein expression has not been widely investigated. We have previously designed a novel 12-mer CPP from the conserved region of the human papillomavirus L1 capsid protein. In this study, we tested whether this peptide, LDP12, effectively delivers a protein cargo to mouse oocytes and preimplantation embryos. We prepared a LDP12–EGFP fusion protein having LDP12 as an N-terminal tag. This fusion protein readily enters HeLa cells

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Tarabarova, A. G., M. S. Yurkova, and A. N. Fedorov. "New eGFP Mutant with Intact C- and N-Termini and Affinity for Ni<sup>2+</sup>." Прикладная биохимия и микробиология 59, no. 6 (2023): 614–21. http://dx.doi.org/10.31857/s0555109923060193.

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The green fluorescent protein GFP has long been used in research practice as a molecular tool. It is often used as a fusion partner. To create fusion constructs, target molecules are attached to the N- or C-terminus of GFP. On the other hand, the N- or C-termini of GFP required to create fusion constructs are also used to attach affinity tags that is greatly facilitating purification. Simultaneous introduction of affinity tag and GFP to both or the same end of GFP can create steric hindrances both in the process of biosynthetic folding of the construct and in its affinity purification. This wo

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8

Jin, Lang, Shahid Mehmood, Giikailang Zhang, et al. "Visualizing Sacbrood Virus of Honey Bees via Transformation and Coupling with Enhanced Green Fluorescent Protein." Viruses 12, no. 2 (2020): 224. http://dx.doi.org/10.3390/v12020224.

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Sacbrood virus (SBV) of honey bees is a picornavirus in the genus Iflavirus. Given its relatively small and simple genome structure, single positive-strand RNA with only one ORF, cloning the full genomic sequence is not difficult. However, adding nonsynonymous mutations to the bee iflavirus clone is difficult because of the lack of information about the viral protein processes. Furthermore, the addition of a reporter gene to the clones has never been accomplished. In preliminary trials, we found that the site between 3′ untranslated region (UTR) and poly(A) can retain added sequences. We added

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WU, Chia-Mao, Hao-Teng CHANG, and Margaret Dah-Tsyr CHANG. "Membrane-bound carboxypeptidase E facilitates the entry of eosinophil cationic protein into neuroendocrine cells." Biochemical Journal 382, no. 3 (2004): 841–48. http://dx.doi.org/10.1042/bj20040894.

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ECP (eosinophil cationic protein) is a major component of eosinophil granule proteins, and is used as a clinical biomarker for asthma and allergic inflammatory disease. ECP has been implicated in damage to the cell membrane of many tissue types, but the mechanism is not well known. In the present study, mECP–eGFP–6H, a recombinant fusion protein containing mature ECP (mECP), enhanced green fluorescence protein (eGFP) and a His6 tag (6H), has been expressed, purified and added to GH3 neuroendocrine cells to study the internalization ability of ECP. We found that mECP–eGFP–6H entered into GH3 ne

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Hamada, Tomohiro, та Yasuo Sakuma. "Estrogen Receptor α Gene Promoter 0/B Usage in the Rat Sexually Dimorphic Nucleus of the Preoptic Area". Endocrinology 151, № 4 (2010): 1923–28. http://dx.doi.org/10.1210/en.2009-1022.

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The volume of the sexually dimorphic nucleus of the preoptic area (SDN-POA) is two to four times larger in male rats than in females; however, the mechanism for the establishment of sexual dimorphism and the function of this nucleus is almost unknown. Perinatal estrogen can cause sexual dimorphism via the estrogen receptor α (ERα). Recently, transgenic rats were generated that express enhanced green fluorescent protein (EGFP) under the control of the ERα gene promoter 0/B to tag ERα-positive neurons in the brain. In the present study, we examined whether this EGFP expression could be a marker

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Müller, Barbara, Jessica Daecke, Oliver T. Fackler, Matthias T. Dittmar, Hanswalter Zentgraf, and Hans-Georg Kräusslich. "Construction and Characterization of a Fluorescently Labeled Infectious Human Immunodeficiency Virus Type 1 Derivative." Journal of Virology 78, no. 19 (2004): 10803–13. http://dx.doi.org/10.1128/jvi.78.19.10803-10813.2004.

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ABSTRACT The introduction of a label which can be detected in living cells opens new possibilities for the direct analysis of dynamic processes in virus replication, such as the transport and assembly of structural proteins. Our aim was to generate a tool for the analysis of the trafficking of the main structural protein of human immunodeficiency virus type 1 (HIV-1), Gag, as well as for the analysis of virus-host cell interactions in an authentic setting. We describe here the construction and characterization of infectious HIV derivatives carrying a label within the Gag polyprotein. Based on

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12

Fang, Yi-Fan, Yi-Lan Li, Xiao-Ming Li, and Ji-Long Liu. "Super-Resolution Imaging Reveals Dynamic Reticular Cytoophidia." International Journal of Molecular Sciences 23, no. 19 (2022): 11698. http://dx.doi.org/10.3390/ijms231911698.

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CTP synthase (CTPS) can form filamentous structures termed cytoophidia in cells in all three domains of life. In order to study the mesoscale structure of cytoophidia, we perform fluorescence recovery after photobleaching (FRAP) and stimulated emission depletion (STED) microscopy in human cells. By using an EGFP dimeric tag as a tool to explore the physical properties of cytoophidia, we find that cytoophidia are dynamic and reticular. The reticular structure of CTPS cytoophidia may provide space for other components, such as IMPDH. In addition, we observe CTPS granules with tentacles.

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Windoffer, R., and R. E. Leube. "Detection of cytokeratin dynamics by time-lapse fluorescence microscopy in living cells." Journal of Cell Science 112, no. 24 (1999): 4521–34. http://dx.doi.org/10.1242/jcs.112.24.4521.

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To monitor the desmosome-anchored cytokeratin network in living cells fusion protein HK13-EGFP consisting of human cytokeratin 13 and the enhanced green fluorescent protein was stably expressed in vulvar carcinoma-derived A-431 cells. It is shown for A-431 subclone AK13-1 that HK13-EGFP emits strong fluorescence in fixed and living cells, being part of an extended cytoplasmic intermediate filament network that is indistinguishable from that of parent A-431 cells. Biochemical, immunological and ultrastructural analyses demonstrate that HK13-EGFP behaves identically to the endogenous cytokeratin

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Lissoni, Alessio, Nan Wang, Timur Nezlobinskii, et al. "Gap19, a Cx43 Hemichannel Inhibitor, Acts as a Gating Modifier That Decreases Main State Opening While Increasing Substate Gating." International Journal of Molecular Sciences 21, no. 19 (2020): 7340. http://dx.doi.org/10.3390/ijms21197340.

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Cx43 hemichannels (HCs) are electrically and chemically gated transmembrane pores with low open probability and multiple conductance states, which makes kinetic studies of channel gating in large datasets challenging. Here, we developed open access software, named HemiGUI, to analyze HC gating transitions and investigated voltage-induced HC opening based on up to ≈4000 events recorded in HeLa-Cx43-overexpressing cells. We performed a detailed characterization of Cx43 HC gating profiles and specifically focused on the role of the C-terminal tail (CT) domain by recording the impact of adding an

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Silin, D., O. Lyubomska, M. Ludlow, W. P. Duprex, and B. K. Rima. "Development of a Challenge-Protective Vaccine Concept by Modification of the Viral RNA-Dependent RNA Polymerase of Canine Distemper Virus." Journal of Virology 81, no. 24 (2007): 13649–58. http://dx.doi.org/10.1128/jvi.01385-07.

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ABSTRACT We demonstrate that insertion of the open reading frame of enhanced green fluorescent protein (EGFP) into the coding sequence for the second hinge region of the viral L (large) protein (RNA-dependent RNA polymerase) attenuates a wild-type canine distemper virus. Moreover, we show that single intranasal immunization with this recombinant virus provides significant protection against challenge with the virulent parental virus. Protection against wild-type challenge was gained either after recovery of cellular immunity postimmunization or after development of neutralizing antibodies. Ins

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Goryacheva, Ekaterina, Roman Efremov, Nikolai Krylov, et al. "Crystal Structure of Bright Fluorescent Protein BrUSLEE with Subnanosecond Fluorescence Lifetime; Electric and Dynamic Properties." International Journal of Molecular Sciences 24, no. 7 (2023): 6403. http://dx.doi.org/10.3390/ijms24076403.

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The rapid development of new microscopy techniques for cell biology has exposed the need for genetically encoded fluorescent tags with special properties. Fluorescent biomarkers of the same color and spectral range and different fluorescent lifetimes (FLs) became useful for fluorescent lifetime image microscopy (FLIM). One such tag, the green fluorescent protein BrUSLEE (Bright Ultimately Short Lifetime Enhanced Emitter), having an extremely short subnanosecond component of fluorescence lifetime (FL~0.66 ns) and exceptional fluorescence brightness, was designed for FLIM experiments. Here, we p

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Usman, Saima, Hebah Aldehlawi, Thuan Nguyen, Muy-Teck Teh, and Ahmad Waseem. "Impact of N-Terminal Tags on De Novo Vimentin Intermediate Filament Assembly." International Journal of Molecular Sciences 23, no. 11 (2022): 6349. http://dx.doi.org/10.3390/ijms23116349.

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Vimentin, a type III intermediate filament protein, is found in most cells along with microfilaments and microtubules. It has been shown that the head domain folds back to associate with the rod domain and this association is essential for filament assembly. The N-terminally tagged vimentin has been widely used to label the cytoskeleton in live cell imaging. Although there is previous evidence that EGFP tagged vimentin fails to form filaments but is able to integrate into a pre-existing network, no study has systematically investigated or established a molecular basis for this observation. To

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Junghans, Cornelia, Franz-Josef Schmitt, Vladana Vukojević, and Thomas Friedrich. "Monitoring the diffusion behavior of Na,K-ATPase by fluorescence correlation spectroscopy (FCS) upon fluorescence labelling with eGFP or Dreiklang." Optofluidics, Microfluidics and Nanofluidics 2, no. 1 (2015): 1–14. http://dx.doi.org/10.1515/optof-2015-0001.

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AbstractMeasurement of lateral mobility of membraneembedded proteins in living cells with high spatial and temporal precision is a challenging task of optofluidics. Biological membranes are complex structures, whose physico-chemical properties depend on the local lipid composition, cholesterol content and the presence of integral or peripheral membrane proteins, which may be involved in supramolecular complexes or are linked to cellular matrix proteins or the cytoskeleton. The high proteinto- lipid ratios in biomembranes indicate that membrane proteins are particularly subject to molecular cro

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AMSTRUP, Jan, and Ivana NOVAK. "P2X7 receptor activates extracellular signal-regulated kinases ERK1 and ERK2 independently of Ca2+ influx." Biochemical Journal 374, no. 1 (2003): 51–61. http://dx.doi.org/10.1042/bj20030585.

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P2X7 nucleotide receptors modulate a spectrum of cellular events in various cells including epithelia, such as exocrine pancreas. Although the pharmacology and channel properties of the P2X7 receptors have been studied intensively, signal transduction pathways are relatively unknown. In this study we applied a heterologous expression system of rat P2X7 receptors in HEK-293 cells. We followed the receptor expression and function using the enhanced green fluorescent protein (EGFP) tag, activation of intracellular proteins and increases in cellular Ca2+. EGFP-P2X7 receptors localized to the plasm

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Ito, Takahito, Akira Suzuki, Enyu Imai, Masaru Okabe, and Masatsugu Hori. "Bone Marrow Is a Reservoir of Repopulating Mesangial Cells during Glomerular Remodeling." Journal of the American Society of Nephrology 12, no. 12 (2001): 2625–35. http://dx.doi.org/10.1681/asn.v12122625.

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ABSTRACT. The renal glomerulus, whose cellular components are developmentally derived from the mesenchyme, plays a pivotal role in filtratating plasma. Irretrievable changes of glomerular components are responsible for the initiation and progression of impaired renal function. Recently, it has been shown that functional stem cells exist in the bone marrow of adult bodies and that they can reconstitute damaged tissues of the mesenchymal origin. To examine whether the bone marrow provides stem cells to damaged glomeruli, transgenic rats carrying enhanced green fluorescence protein (EGFP rat) wer

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Haqshenas, G., J. M. Mackenzie, X. Dong, and E. J. Gowans. "Hepatitis C virus p7 protein is localized in the endoplasmic reticulum when it is encoded by a replication-competent genome." Journal of General Virology 88, no. 1 (2007): 134–42. http://dx.doi.org/10.1099/vir.0.82049-0.

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p7 protein is a small protein encoded by Hepatitis C virus (HCV) that functions as an ion channel in planar lipid bilayers. The function of p7 is vital for the virus life cycle. In this study, the p7 protein of genotype 2a (strain JFH1; the only strain that replicates and produces virus progeny in vitro) was tagged with either an enhanced green fluorescent protein (eGFP) or a haemagglutinin (HA) epitope to facilitate tracking of the protein in the intracellular environment. The tagged viral polyprotein was expressed transiently in the cells after transfection with the recombinant RNA transcrip

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Kulichkova, Valentina A., Tatiana O. Artamonova, Julia J. Zaykova та ін. "Simultaneous EGFP and Tag Labeling of the β7 Subunit for Live Imaging and Affinity Purification of Functional Human Proteasomes". Molecular Biotechnology 57, № 1 (2014): 36–44. http://dx.doi.org/10.1007/s12033-014-9799-0.

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Kulichkova, V. A., Yu Ya Zaykova, Yu B. Ermolaeva, et al. "Creation of a HEK293 cell line stably expressing the proteasome subunit PSMD14 fused with fluorescent protein EGFP and HTBT tag." Cell and Tissue Biology 8, no. 4 (2014): 330–36. http://dx.doi.org/10.1134/s1990519x14040051.

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Shibata, Toshikatsu, Kuniaki Nerome, Mitsuhiko Moriyama, Satoshi Hayakawa, and Kazumichi Kuroda. "Addition of an EGFP-tag to the N-terminal of influenza virus M1 protein impairs its ability to accumulate in ND10." Journal of Virological Methods 252 (February 2018): 75–79. http://dx.doi.org/10.1016/j.jviromet.2017.11.008.

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García-Martínez, Cèlia, Mario Marotta, Rodrigo Moore-Carrasco, et al. "Impact on fatty acid metabolism and differential localization of FATP1 and FAT/CD36 proteins delivered in cultured human muscle cells." American Journal of Physiology-Cell Physiology 288, no. 6 (2005): C1264—C1272. http://dx.doi.org/10.1152/ajpcell.00271.2004.

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We compared the intracellular distribution and regulatory role of fatty acid transporter protein (FATP1) and fatty acid translocase (FAT/CD36) on muscle cell fatty acid metabolism. With the use of adenoviruses, FATP1 and FAT genes were delivered to primary cultured human muscle cells. FATP1 and FAT moderately enhanced palmitate and oleate transport evenly at concentrations of 0.05, 0.5, and 1 mM. Long-term (16 h) consumption of palmitate and oleate from the media, and particularly incorporation into triacylglyceride (TAG), was stimulated equivalently by FATP1 and FAT at all fatty acid concentr

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Shiraishi, Ryo, Gabriele Cancila, Kohei Kumegawa, et al. "MEDB-15. Dynamic chromatin alteration induces oncogenic hijacking by essential transcriptional factors during SHH medulloblastoma tumorigenesis." Neuro-Oncology 24, Supplement_1 (2022): i107—i108. http://dx.doi.org/10.1093/neuonc/noac079.390.

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Abstract Medulloblastoma is a malignant brain tumor that occurs in the cerebellum, most frequently in children. Medulloblastoma is molecularly classified into four major groups, and therapies are now being developed according to the nature of these groups and subgroups. However, there are currently no effective molecularly targeted drugs for most of these groups. In recent years, we have been analyzing the genomes of medulloblastomas to identify genetic mutations involved in tumorigenesis. Among them, mutations in chromatin modifiers are frequently detected in medulloblastoma, suggesting the i

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Kudo, Ken-ichi, Naohiro Tsuyama, Misaki Takahashi Sugai, et al. "Normal B Cell-Derived iPSCs Capable of Inducing RAS Mutants and Aid to Explore Myeloma-Initiating Cells." Blood 138, Supplement 1 (2021): 4711. http://dx.doi.org/10.1182/blood-2021-149108.

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Abstract Multiple myeloma (MM) cells are derived from mature B cells based on immunoglobulin heavy chain (IgH) gene analysis. The onset of MM is often caused by a reciprocal chromosomal translocation (cTr) between chr 14 with IgH and chr 11 with CCND1. We propose that mature B cells gain the potential to transform by reprograming, and the resulting chromosomal aberrations subsequently cause the development of abnormal B cells as a myeloma-initiating cell during B cell redifferentiation. To study myeloma-initiating cells, we previously established normal B cell-derived induced pluripotent stem

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Ren, Kehan, Zongjun Xia, Ermin Li, Xu Han, and Peng Ji. "Rapid Degradation of mDia2 Protein during Terminal Erythropoiesis Via an In Vivo Aid System: An Alternative Approach for Loss-of-Function Studies." Blood 142, Supplement 1 (2023): 2443. http://dx.doi.org/10.1182/blood-2023-178268.

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Loss-of-function manipulations are crucial methods for studying gene functions. Despite the availability of many chemical-induced genetic manipulation techniques, there are limitations associated with these technologies. Tamoxifen-induced conditional gene expression, gene knockdown, or cell tracking represent some of the most common genetic manipulations employed in mice. However, tamoxifen-induced gene or Cre expression in bone marrow cell populations is relatively weak, affecting only a low proportion of cells. Similar observations have been made with the in vivo doxycycline-inducible system

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ZHANG, Wenzheng, Yoshihide HAYASHIZAKI, and Bruce C. KONE. "Structure and regulation of the mDot1 gene, a mouse histone H3 methyltransferase." Biochemical Journal 377, no. 3 (2004): 641–51. http://dx.doi.org/10.1042/bj20030839.

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Recently, a new class of histone methyltransferases that plays an indirect role in chromatin silencing by targeting a conserved lysine residue in the nucleosome core was described, namely the Dot1 (disruptor of telomeric silencing) family [Feng, Wang, Ng, Erdjument-Bromage, Tempst, Struhl and Zhang (2002) Curr. Biol. 12, 1052–1058; van Leeuwen, Gafken and Gottschling (2002) Cell (Cambridge, Mass.) 109, 745–756; Ng, Feng, Wang, Erdjument-Bromage, Tempst, Zhang and Struhl (2002) Genes Dev. 16, 1518–1527]. In the present study, we report the isolation, genomic organization and in vivo expression

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Bannach, Carina, Daniel Ruiz Buck, Genna Bobby, et al. "Optimizing Recombinant Baculovirus Vector Design for Protein Production in Insect Cells." Processes 9, no. 12 (2021): 2118. http://dx.doi.org/10.3390/pr9122118.

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Autographa californica nucleopolyhedrovirus is a very productive expression vector for recombinant proteins in insect cells. Most vectors are based on the polyhedrin gene promoter, which comprises a TAAG transcription initiation motif flanked by 20 base pairs upstream and 47 base pairs downstream before the native ATG. Many transfer vectors also include a short sequence downstream of the ATG, in which case this sequence is mutated to ATT to abolish translation. However, the ATT sequence, or AUU in the mRNA, is known to be leaky. If a target-coding region is placed in the frame with the AUU, th

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Schopf, Krystina, Thomas K. Smylla, and Armin Huber. "Immunocytochemical Labeling of Rhabdomeric Proteins inDrosophilaPhotoreceptor Cells Is Compromised by a Light-dependent Technical Artifact." Journal of Histochemistry & Cytochemistry 67, no. 10 (2019): 745–57. http://dx.doi.org/10.1369/0022155419859870.

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Drosophila photoreceptor cells are employed as a model system for studying membrane protein transport. Phototransduction proteins like rhodopsin and the light-activated TRPL ion channel are transported within the photoreceptor cell, and they change their subcellular distribution in a light-dependent way. Investigating the transport mechanisms for rhodopsin and ion channels requires accurate histochemical methods for protein localization. By using immunocytochemistry the light-triggered translocation of TRPL has been described as a two-stage process. In stage 1, TRPL accumulates at the rhabdome

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Tao, Yu, Gaojian Li, Wenqian Zheng, et al. "Development of a Combined Genetic Engineering Vaccine for Porcine Circovirus Type 2 and Mycoplasma Hyopneumoniae by a Baculovirus Expression System." International Journal of Molecular Sciences 20, no. 18 (2019): 4425. http://dx.doi.org/10.3390/ijms20184425.

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Mycoplasma hyopneumoniae (Mhp) and porcine circovirus type 2 (PCV2) are the main pathogens for mycoplasmal pneumonia of swine (MPS) and post-weaning multisystemic wasting syndrome (PMWS), respectively. Infection by these pathogens often happens together and causes great economic losses. In this study, a kind of recombinant baculovirus that can display P97R1P46P42 chimeric protein of Mhp and the capsid (Cap) protein of PCV2 was developed, and the protein location was identified. Another recombinant baculovirus was constructed without tag proteins (EGFP, mCherry) and was used to evaluate the imm

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Beckers, Constantin J., Achmed Mrestani, Fabian Komma, and Sven Dannhäuser. "Versatile Endogenous Editing of GluRIIA in Drosophila melanogaster." Cells 13, no. 4 (2024): 323. http://dx.doi.org/10.3390/cells13040323.

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Glutamate receptors at the postsynaptic side translate neurotransmitter release from presynapses into postsynaptic excitation. They play a role in many forms of synaptic plasticity, e.g., homeostatic scaling of the receptor field, activity-dependent synaptic plasticity and the induction of presynaptic homeostatic potentiation (PHP). The latter process has been extensively studied at Drosophila melanogaster neuromuscular junctions (NMJs). The genetic removal of the glutamate receptor subunit IIA (GluRIIA) leads to an induction of PHP at the synapse. So far, mostly imprecise knockouts of the Glu

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Shen, Yihan, Nagendra Babu Thillaiappan, and Colin W. Taylor. "The store-operated Ca2+ entry complex comprises a small cluster of STIM1 associated with one Orai1 channel." Proceedings of the National Academy of Sciences 118, no. 10 (2021): e2010789118. http://dx.doi.org/10.1073/pnas.2010789118.

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Increases in cytosolic Ca2+ concentration regulate diverse cellular activities and are usually evoked by opening of Ca2+ channels in intracellular Ca2+ stores and the plasma membrane (PM). For the many signals that evoke formation of inositol 1,4,5-trisphosphate (IP3), IP3 receptors coordinate the contributions of these two Ca2+ sources by mediating Ca2+ release from the endoplasmic reticulum (ER). Loss of Ca2+ from the ER then activates store-operated Ca2+ entry (SOCE) by causing dimers of STIM1 to cluster and unfurl cytosolic domains that interact with the PM Ca2+ channel, Orai1, causing its

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Wouters, Mira, Karine Smans, and Jean-Marie Vanderwinden. "WZsGreen/+: a new green fluorescent protein knock-in mouse model for the study of KIT-expressing cells in gut and cerebellum." Physiological Genomics 22, no. 3 (2005): 412–21. http://dx.doi.org/10.1152/physiolgenomics.00105.2005.

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In the small intestine, interstitial cells of Cajal (ICC) surrounding the myenteric plexus generate the pacemaking slow waves that are essential for an efficient intestinal transit. The underlying molecular mechanisms of the slow wave are poorly known. KIT is currently the sole practical marker for ICC. Attempts to purify living ICC have so far largely failed, due to the loss of the KIT epitope during enzymatic dissociation. Aiming to identify and isolate living ICC, we designed a knock-in strategy to express a fluorescent tag in KIT-expressing cells by inserting the sequence of the novel gree

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Yarinich, L. A., A. A. Ogienko, A. V. Pindyurin, and E. S. Omelina. "Analysis of the transcriptional activity of model piggyBac transgenes stably integrated into different loci of the genome of CHO cells in the absence of selection pressure." Vavilov Journal of Genetics and Breeding 27, no. 7 (2023): 906–15. http://dx.doi.org/10.18699/vjgb-23-105.

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CHO cells are most commonly used for the synthesis of recombinant proteins in biopharmaceutical production. When stable producer cell lines are obtained, the locus of transgene integration into the genome has a great influence on the level of its expression. Therefore, the identification of genomic loci ensuring a high level of protein production is very important. Here, we used the TRIP assay to study the influence of the local chromatin environment on the activity of transgenes in CHO cells. For this purpose, reporter constructs encoding eGFP under the control of four promoters were stably i

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Taylor, Samuel, Jacob Stauber, Oliver Bohorquez, et al. "Transcription Factor Redistributors Pharmacologically Actuate Non-Canonical Gene Networks to Drive AML Differentiation." Blood 142, Supplement 1 (2023): 119. http://dx.doi.org/10.1182/blood-2023-186698.

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Aberrant transcriptional networks are hallmarks of aging and cancer, yet our ability to target these aberrations is poor. PU.1 is one such transcription factor (TF) who's transcriptional networks are corrupted in disease, including in &gt;50% of Acute Myeloid Leukemia (AML) cases. In this study we investigate an unappreciated pharmacological approach to target the aberrant PU.1 network, by employing novel small molecules which competitively inhibit PU.1:DNA interactions. We performed an extensive multiomics-driven, molecular characterization of AML cells following exposure to PU.1-DNA bind

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Yuan, Ping, Sirisha M. Cheedipudi, Leila Rouhi, et al. "Single-Cell RNA Sequencing Uncovers Paracrine Functions of the Epicardial-Derived Cells in Arrhythmogenic Cardiomyopathy." Circulation 143, no. 22 (2021): 2169–87. http://dx.doi.org/10.1161/circulationaha.120.052928.

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Background: Arrhythmogenic cardiomyopathy (ACM) manifests with sudden death, arrhythmias, heart failure, apoptosis, and myocardial fibro-adipogenesis. The phenotype typically starts at the epicardium and advances transmurally. Mutations in genes encoding desmosome proteins, including DSP (desmoplakin), are major causes of ACM. Methods: To delineate contributions of the epicardium to the pathogenesis of ACM, the Dsp allele was conditionally deleted in the epicardial cells in mice upon expression of tamoxifen-inducible Cre from the Wt1 locus. Wild type (WT) and Wt1-Cre ERT2 :Dsp W/F were crossed

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Azad, Md Thoufic A., Umme Qulsum, and Toshifumi Tsukahara. "Comparative Activity of Adenosine Deaminase Acting on RNA (ADARs) Isoforms for Correction of Genetic Code in Gene Therapy." Current Gene Therapy 19, no. 1 (2019): 31–39. http://dx.doi.org/10.2174/1566523218666181114122116.

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Introduction: Members of the adenosine deaminase acting on RNA (ADAR) family of enzymes consist of double-stranded RNA-binding domains (dsRBDs) and a deaminase domain (DD) that converts adenosine (A) into inosine (I), which acts as guanosine (G) during translation. Using the MS2 system, we engineered the DD of ADAR1 to direct it to a specific target. The aim of this work was to compare the deaminase activities of ADAR1-DD and various isoforms of ADAR2-DD. Materials and Methods: We measured the binding affinity of the artificial enzyme system on a Biacore ™ X100. ADARs usually target dsRNA, so

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Tan, Xiao-Jun, Xiao-Wei Xing, Lu-Yun Li, et al. "Molecular Cloning of a Novel Mouse Testis-specific Spermatogenic Cell Apoptosis Inhibitor Gene mTSARG7 as a Candidate Oncogene." Acta Biochimica et Biophysica Sinica 37, no. 6 (2005): 396–405. http://dx.doi.org/10.1111/j.1745-7270.2005.00057.x.

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Abstract A novel mouse gene, mTSARG7 (GenBank accession No. AY489184), with a full cDNA length of 2279 bp and containing 12 exons and 11 introns, was cloned from a mouse expressed sequence tag (GenBank accession No. BE644543) that was significantly up-regulated in cryptorchidism. The gene was located in mouse chromosome 8A1.3 and encoded a protein containing 403 amino acid residues that was a new member of the acyltransferase family because the sequence contained the highly conserved phosphate acyltransferase (PlsC) domain existing in all acyltransferase-like proteins. The mTSARG7 protein and

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Buchner, David A., Jordan A. Shavit, Fengyun Su, et al. "pak2a Mutations Cause Cerebral Hemorrhage in Redhead Zebrafish." Blood 108, no. 11 (2006): 142. http://dx.doi.org/10.1182/blood.v108.11.142.142.

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Abstract Zebrafish are a powerful vertebrate model system for the study of human disease as they share many molecular pathways with mammals. Of note, nearly all of the mammalian coagulation factors are also highly conserved in fish. As part of a whole genome ENU mutagenesis screen, we identified a mutant zebrafish which displayed intraventricular hemorrhage between 2–3 days post fertilization (dpf), which we named redhead. Using an F2 intercross strategy, we mapped this recessive mutant to a 100 kilobase interval on chromosome 2 and identified a splice site mutation in the gene for an ortholog

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Nasri, Masoud, Perihan Mir, Benjamin Dannenmann, et al. "A Method to Fluorescently Label the CRISPR/Cas9-gRNA RNP Complexes Enables Enrichment of Clinical-Grade Gene-Edited Primary Hematopoietic Stem Cells and iPSCs." Blood 132, Supplement 1 (2018): 1108. http://dx.doi.org/10.1182/blood-2018-99-114844.

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Abstract Although proven to be an excellent method for gene editing, CRISPR/Cas9-mediated technology still has some limitations for the applications in primary hematopoietic stem cells and progenitor cells (HSPCs) as well as in human induced pluripotent stem cells (hiPSCs). Delivery of Cas9 protein in a form of ribonucleoprotein (RNP) in a complex with guide RNA (gRNA) provides a DNA free methodology, but a big hinderance of this application is that it is not possible to sort and enrich gene edited cells for further applications. Here we report the establishment of a new protocol of fluorescen

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Wu, Junxian, Weiwei Liu, Jimei Lu, Rui Xu, Jin Xie, and Liangping Zha. "Cloning, Prokaryotic Expression, and Purification of Acetyl-CoA C-Acetyltransferase from Atractylodes lancea." Protein & Peptide Letters 29, no. 2 (2022): 156–65. http://dx.doi.org/10.2174/0929866528666211126162838.

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Background: Cangzhu (Atractylodes lancea), a valuable and common traditional Chinese medicinal herb, is primarily used as an effective medicine with various health-promoting effects. The main pharmacological bioactive ingredients in the rhizome of A. lancea are terpenoids. Acetyl-CoA C-acetyltransferase (AACT) is the first enzyme in the terpenoid synthesis pathway and catalyzes two units of acetyl-CoA into acetoacetyl-CoA. Objective: The objective of the present work was to clone and identify function of AlAACT from Atractylodes lancea. Method: A full-length cDNA clone of AlAACT was isolated u

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Kuo, Pei-Yu, Zewei Jiang, Deepak Perumal, et al. "SOX11 Cooperates with CCND1 in Mantle Cell Lymphoma Pathogenesis." Blood 126, no. 23 (2015): 1253. http://dx.doi.org/10.1182/blood.v126.23.1253.1253.

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Abstract MCL (Mantle cell lymphoma) is an aggressive and incurable B cell malignancy with a median survival of 5-6 years. Cyclin D1 (CCND1) overexpression is a key diagnostic feature of this disease, observed in more than 90% of MCL tumors. However, murine models over-expressing CCND1 in B cells do not recapitulate the phenotype of MCL. The SOX11 transcription factor is aberrantly expressed in 80-90% of primary MCL. Our published data demonstrated that SOX11 binds and functionally regulates key components in multiple oncogenic pathways in MCL such as WNT and TGFβ pathways. Recent studies have

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Strachotová, Dita, Aleš Holoubek, Barbora Brodská, and Petr Herman. "Two-photon lifetime-Based Photoconversion of EGFP for 3D-photostimulation in FLIM." Methods and Applications in Fluorescence, June 2, 2023. http://dx.doi.org/10.1088/2050-6120/acdb31.

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Abstract Enhanced green fluorescence protein (EGFP) is a fluorescent tag commonly used in cellular and biomedical applications. Surprisingly, some interesting photochemical properties of EGFP have remained unexplored. Here we report on two-photon-induced photoconversion of EGFP, which can be permanently converted by intense IR irradiation to a form with a short fluorescence lifetime and spectrally conserved emission. Photoconverted EGFP thus can be distinguished from the unconverted tag by the time-resolved detection. Nonlinear dependence of the two-photon photoconversion efficiency on the lig

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Letra-Vilela, Ricardo, Ricardo Quiteres, Fernanda Murtinheira, Alvaro Crevenna, Zach Hensel, and Federico Herrera. "New tools for the visualization of glial fibrillary acidic protein in living cells." Experimental Results 1 (2020). http://dx.doi.org/10.1017/exp.2020.1.

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AbstractThe glial fibrillary acidic protein (GFAP) is an intermediate filament widely used to identify and label astroglial cells, a very abundant and relevant glial cell type in the central nervous system. A major hurdle in studying its behavior and function arises from the fact that GFAP does not tolerate well the addition of protein tags to its termini. Here, we tagged human GFAP (hGFAP) with an enhanced green fluorescent protein (EGFP) for the first time, and substituted a previously reported EGFP tag on mouse GFAP (mGFAP) by a more versatile Halo Tag. Both versions of tagged GFAP were abl

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Yang, Lele, Lifang Cui, Shumin Ma, Qingqing Zuo, and Qilai Huang. "A Gene Transfer-Positive Cell Sorting System Utilizing Membrane-Anchoring Affinity Tag." Frontiers in Bioengineering and Biotechnology 10 (June 16, 2022). http://dx.doi.org/10.3389/fbioe.2022.930966.

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Gene delivery efficiency is an essential limit factor in gene study and gene therapy, especially for cells that are hard for gene transfer. Here we develop an affinity cell sorting system that allows efficient enrichment of gene transfer-positive cells. The system expresses an enhanced green fluorescent protein (EGFP) fused with an N-terminal high-affinity Twin-Strep-Tag (TST) that will be anchored to the cell membrane at the out-surface through a glycosylphosphatidylinositol (GPI) membrane-anchoring structure. The EGFP permits microscopy and flow cytometry analysis of the gene transfer-positi

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Nørgård, Mikkel Ø., Lasse B. Steffensen, Didde R. Hansen, et al. "A new transgene mouse model using an extravesicular EGFP tag enables affinity isolation of cell-specific extracellular vesicles." Scientific Reports 12, no. 1 (2022). http://dx.doi.org/10.1038/s41598-021-04512-0.

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AbstractThe in vivo function of cell-derived extracellular vesicles (EVs) is challenging to establish since cell-specific EVs are difficult to isolate and differentiate. We, therefore, created an EV reporter using truncated CD9 to display enhanced green fluorescent protein (EGFP) on the EV surface. CD9truc-EGFP expression in cells did not affect EV size and concentration but enabled co-precipitation of EV markers TSG101 and ALIX from the cell-conditioned medium by anti-GFP immunoprecipitation. We then created a transgenic mouse where CD9truc-EGFP was inserted in the inverse orientation and dou

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Gao, Jianhua, Chunping Ouyang, Juanli Zhao, et al. "Coexpressing the Signal Peptide of Vip3A and the Trigger Factor of Bacillus thuringiensis Enhances the Production Yield and Solubility of eGFP in Escherichia coli." Frontiers in Microbiology 13 (July 18, 2022). http://dx.doi.org/10.3389/fmicb.2022.892428.

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Many fusion tags have been developed to improve the expression of recombinant proteins. Besides the translocation of cargo proteins, the signal peptides (SPs) of some secretory proteins, such as the ssTorA and Iasp, have been used as an inclusion body tag (IB-tag) or the recombinant expression enhancer in the cytosol of E. coli. In this study, the approach to utilize the SP of Vip3A (Vasp) from Bacillus thuringiensis (Bt) as a fusion tag was investigated. The results showed that either the Vasp or its predicted N- (VN), H- (VH), and C-regions (VC), as well as their combinations (VNH, VNC, and

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Li, Wencheng, and Yumei Feng. "Abstract 079: (pro)renin Receptor Knockdown In The Paraventricular Nucleus Of Hypothalamus Attenuates The Development Of Doca-salt Hypertension." Hypertension 64, suppl_1 (2014). http://dx.doi.org/10.1161/hyp.64.suppl_1.079.

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We previously reported that neuron-specific PRR deletion prevents the development of deoxycorticosterone acetate (DOCA)-salt induced hypertension. However, it is not clear which brain region is responsible for the effect of PRR knockout on the development of hypertension. In present study, we bilaterally micro-injected adeno-associated virus (AAV2)-mediated Cre recombinase with an eGFP tag (AAV-Cre, 50 nl/side) into the paraventricular nucleus of hypothalamus (PVN) of PRR-Floxed mice to knockdown the PRR expression in this region. Deoxycorticosterone acetate-salt (DOCA, 50mg, 0.9%NaCl drinking

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