DOCSLIB.ORG

Mouse Ern1 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Ern1 Conditional Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Ern1 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Ern1 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Ern1 gene ( NCBI Reference Sequence: NM_023913 ; Ensembl: ENSMUSG00000020715 ) is located on mouse chromosome 11. 22 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 22 (Transcript: ENSMUST00000001059). Exon 2 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Ern1 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-386O11 as template. Cas9, gRNA and targeting vector will be co-injected into fertilized eggs for cKO mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for null mutations display embryonic lethality during organogenesis. Homozygous mice may also display decreased embryo size, impaired hematopoiesis, and/or placental abnormalities. The knockout of Exon 2 will result in frameshift of the gene, and covers 4.13% of the coding region. The size of intron 1 for 5'-loxP site insertion: 28588 bp, and the size of intron 2 for 3'-loxP site insertion: 21784 bp. The size of effective cKO region: ~1620 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. Page 1 of 7 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 22 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Ern1 Homology arm cKO region loxP site Page 2 of 7 http://www.alphaknockout.com/ Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7121bp) | A(26.63% 1896) | C(19.59% 1395) | G(22.29% 1587) | T(31.5% 2243) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 7 http://www.alphaknockout.com/ BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr11 - 106459279 106462278 3000 browser details YourSeq 213 2505 2885 3000 90.3% chr4 + 132903419 132903766 348 browser details YourSeq 197 2503 2886 3000 89.9% chr16 + 18438940 18439256 317 browser details YourSeq 190 2677 2902 3000 95.8% chr5 - 121456971 121457210 240 browser details YourSeq 190 2526 2885 3000 96.7% chr11 - 96990726 96991258 533 browser details YourSeq 190 2672 2885 3000 93.0% chr4 + 123761529 123761729 201 browser details YourSeq 187 2678 2887 3000 97.5% chr6 - 34898766 34898981 216 browser details YourSeq 187 2676 2885 3000 96.6% chr3 - 121963381 121963591 211 browser details YourSeq 187 2683 2882 3000 96.0% chr2 + 97575334 97575532 199 browser details YourSeq 187 2673 2884 3000 97.0% chr18 + 4058595 4058813 219 browser details YourSeq 186 2681 2885 3000 98.5% chr12 - 111472406 111472617 212 browser details YourSeq 186 2677 2901 3000 95.2% chr17 + 22366064 22366288 225 browser details YourSeq 185 2678 2885 3000 93.5% chr8 - 117428076 117428273 198 browser details YourSeq 185 2678 2884 3000 97.5% chr15 - 82751079 82751295 217 browser details YourSeq 185 2678 2884 3000 93.9% chr19 + 53763673 53763868 196 browser details YourSeq 184 2678 2884 3000 97.5% chrX - 82253604 82253817 214 browser details YourSeq 184 2678 2884 3000 97.5% chr9 - 56245554 56245768 215 browser details YourSeq 184 2695 2901 3000 96.1% chr9 - 20392946 20393154 209 browser details YourSeq 184 2678 2885 3000 97.5% chr7 - 28707963 28708205 243 browser details YourSeq 184 2682 2885 3000 98.0% chr17 - 70359064 70359273 210 Note: The 3000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr11 - 106455658 106458657 3000 browser details YourSeq 255 375 694 3000 91.3% chr2 + 128725450 128725782 333 browser details YourSeq 247 375 698 3000 93.4% chr3 + 57615221 57615587 367 browser details YourSeq 246 373 689 3000 87.6% chr7 + 110783773 110784078 306 browser details YourSeq 245 378 692 3000 90.5% chr8 - 22473861 22474184 324 browser details YourSeq 244 367 694 3000 88.8% chr15 - 67511609 67511946 338 browser details YourSeq 243 378 711 3000 91.3% chr5 - 120765402 120765756 355 browser details YourSeq 243 378 690 3000 91.4% chr8 + 117289757 117290091 335 browser details YourSeq 243 375 689 3000 92.7% chr3 + 97218513 97218851 339 browser details YourSeq 242 369 715 3000 90.9% chr16 + 32450411 32450775 365 browser details YourSeq 241 378 694 3000 93.3% chrX - 153677965 153678311 347 browser details YourSeq 241 377 694 3000 86.9% chr17 - 74505541 74505848 308 browser details YourSeq 241 375 694 3000 90.8% chr13 - 8997205 8997727 523 browser details YourSeq 241 375 706 3000 89.6% chrX + 94024090 94024414 325 browser details YourSeq 241 375 694 3000 90.9% chr3 + 103162503 103162863 361 browser details YourSeq 240 376 694 3000 89.7% chr9 - 75433618 75433965 348 browser details YourSeq 240 375 676 3000 91.4% chr16 - 11163008 11163318 311 browser details YourSeq 240 377 694 3000 88.3% chr15 - 83554404 83554716 313 browser details YourSeq 240 379 690 3000 89.9% chr14 + 20353783 20354089 307 browser details YourSeq 239 376 690 3000 88.0% chr18 - 64880049 64880356 308 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 http://www.alphaknockout.com/ Gene and protein information: Ern1 endoplasmic reticulum (ER) to nucleus signalling 1 [ Mus musculus (house mouse) ] Gene ID: 78943, updated on 10-Jan-2021 Gene summary Official Symbol Ern1 provided by MGI Official Full Name endoplasmic reticulum (ER) to nucleus signalling 1 provided by MGI Primary source MGI:MGI:1930134 See related Ensembl:ENSMUSG00000020715 Gene type protein coding RefSeq status PROVISIONAL Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as Ir; Ire; Ire1a; Ire1p; C85377; AI225830; Ire1alpha; 9030414B18Rik Expression Broad expression in adrenal adult (RPKM 76.6), ovary adult (RPKM 25.1) and 15 other tissues See more Orthologs human all Genomic context Location: 11; 11 E1 See Ern1 in Genome Data Viewer Exon count: 22 Annotation release Status Assembly Chr Location 109 current GRCm39 (GCF_000001635.27) 11 NC_000077.7 (106285474..106378701, complement) 108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (106397620..106487796, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (106258934..106349110, complement) Chromosome 11 - NC_000077.7 Page 5 of 7 http://www.alphaknockout.com/ Transcript information: This gene has 5 transcripts Gene: Ern1 ENSMUSG00000020715 Description endoplasmic reticulum (ER) to nucleus signalling 1 [Source:MGI Symbol;Acc:MGI:1930134] Gene Synonyms 9030414B18Rik, Ire1a, Ire1alpha, Ire1p Location Chromosome 11: 106,394,650-106,487,852 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 195 orthologues, 1 paralogue and is associated with 15 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Match Flags Ern1- ENSMUST00000001059.8 6946 977aa ENSMUSP00000001059.2 Protein coding CCDS25556 Q9EQY0 TSL:1 201 GENCODE basic APPRIS P1 Ern1- ENSMUST00000106801.7 1957 408aa ENSMUSP00000102413.1 Protein coding - Q3TBZ5 TSL:1 204 Q9EQY0 GENCODE basic Ern1- ENSMUST00000106799.1 660 66aa ENSMUSP00000102411.1 Protein coding - B1ATQ7 TSL:1 202 GENCODE basic Ern1- ENSMUST00000106800.1 654 118aa ENSMUSP00000102412.1 Protein coding - B1ARJ5 TSL:3 203 GENCODE basic Ern1- ENSMUST00000131895.1 3354 No - Processed - - TSL:5 205 protein transcript 113.20 kb Forward strand 106.40Mb 106.42Mb 106.44Mb 106.46Mb 106.48Mb Contigs AL604045.7 > AL663053.12 > Genes (Comprehensive set... < Icam2-203protein coding < Ern1-204protein coding < Icam2-204protein coding < Ern1-203protein coding < Ern1-201protein coding < Ern1-205processed transcript < Ern1-202protein coding Regulatory Build 106.40Mb 106.42Mb 106.44Mb 106.46Mb 106.48Mb Reverse strand 113.20 kb Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding processed transcript Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Page 6 of 7 http://www.alphaknockout.com/ Transcript: ENSMUST00000001059 < Ern1-201protein
Load more

Recommended publications
  • Ire1β Negatively Regulates Ire1α Signaling in Response to Endoplasmic Reticulum Stress
    bioRxiv preprint doi: https://doi.org/10.1101/586305; this version posted March 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. IRE1 negatively regulates IRE1 signaling in response to endoplasmic reticulum stress Michael J. Grey1,2,3†, Eva Cloots4,5†, Mariska S. Simpson1,6†, Nicole LeDuc1, Yevgeniy V. Serebrenik7, Heidi De Luca1, Delphine De Sutter4, Phi Luong1, Jay R. Thiagarajah1,2,3, Adrienne W. Paton8, James C. Paton8, Markus A. Seeliger9, Sven Eyckerman4, Sophie Janssens5, and Wayne I. Lencer1,2,3* 1Division of Gastroenterology, Hepatology, and Nutrition, Boston Children’s Hospital, Boston, MA 02115 2Harvard Medical School, Boston MA 02115 3Harvard Digestive Disease Center, Boston MA 02115 4VIB-UGent Center for Medical Biotechnology and Department of Biomolecular Medicine, Ghent University, Ghent, Belgium 5Laboratory for ER stress and Inflammation, VIB-UGent Center for Inflammation Research and Department of Internal Medicine and Pediatrics, Ghent University, Ghent, Belgium 6Molecular and Cellular Sciences, Graduate School of Life Sciences, Utrecht University, Utrecht, The Netherlands 7Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511 8Research Centre for Infectious Diseases, Department of Molecular and Biomedical Science, University of Adelaide, Adelaide, SA, Australia 9Department of Pharmacological Sciences, Stony Brook University Medical School, Stony Brook, NY 17794 †These authors contributed equally to this work. 1 bioRxiv preprint doi: https://doi.org/10.1101/586305; this version posted March 23, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.
    [Show full text]
  • ERN1 Knockdown Modifies the Impact of Glucose and Glutamine Deprivations on the Expression of EDN1 and Its Receptors in Glioma Cells
    This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/ by-nc-nd/4.0), which permits copy and redistribute the material in any medium or format, provided the original work is properly cited. 72 ENDOCRINE REGULATIONS, Vol. 55, No. 2, 72–82, 2021 doi:10.2478/enr-2021-0009 ERN1 knockdown modifies the impact of glucose and glutamine deprivations on the expression of EDN1 and its receptors in glioma cells Dmytro O. Minchenko1,2, Olena O. Khita1, Dariia O. Tsymbal1, Yuliia M. Viletska1, Myroslava Y. Sliusar1, Yuliia V. Yefimova1, Liudmyla O. Levadna2, Dariia A. Krasnytska1, Oleksandr H. Minchenko1 1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2National Bogomolets Medical University, Kyiv, Ukraine E-mail: [email protected] Objective. The aim of the present investigation was to study the impact of glucose and gluta- mine deprivations on the expression of genes encoding EDN1 (endothelin-1), its cognate receptors (EDNRA and EDNRB), and ECE1 (endothelin converting enzyme 1) in U87 glioma cells in re- sponse to knockdown of ERN1 (endoplasmic reticulum to nucleus signaling 1), a major signaling pathway of endoplasmic reticulum stress, for evaluation of their possible implication in the control of glioma growth through ERN1 and nutrient limitations. Methods. The expression level of EDN1, its receptors and converting enzyme 1 in control U87 glioma cells and cells with knockdown of ERN1 treated by glucose or glutamine deprivation by quantitative polymerase chain reaction was studied. Results. We showed that the expression level of EDN1 and ECE1 genes was significantly up- regulated in control U87 glioma cells exposure under glucose deprivation condition in compari- son with the glioma cells, growing in regular glucose containing medium.
    [Show full text]
  • A Human Genome-Wide Rnai Screen Reveals Diverse Modulators That Mediate Ire1a–XBP1 Activation Zhifen Yang1, Jing Zhang1, Dadi Jiang2, Purvesh Khatri3, David E
    Published OnlineFirst February 9, 2018; DOI: 10.1158/1541-7786.MCR-17-0307 Molecular Cancer Research A Human Genome-Wide RNAi Screen Reveals Diverse Modulators that Mediate IRE1a–XBP1 Activation Zhifen Yang1, Jing Zhang1, Dadi Jiang2, Purvesh Khatri3, David E. Solow-Cordero4, Diego A.S. Toesca1, Constantinos Koumenis5, Nicholas C. Denko6, Amato J. Giaccia1, Quynh-Thu Le1, and Albert C. Koong2 Abstract Activation of the unfolded protein response (UPR) signaling pathways is linked to multiple human diseases, including cancer. The inositol-requiring kinase 1a (IRE1a)–X-box binding protein 1 (XBP1) pathway is the most evo- lutionarily conserved of the three major signaling branches of the UPR. Here, we performed a genome-wide siRNA screen to obtain a systematic assessment of genes integrated in the IRE1a– XBP1 axis. We monitored the expression of an XBP1-luciferase chimeric protein in which lucifer- ase was fused in-frame with the spliced (active) form of XBP1. Using cellsexpressingthis reporter construct, we identified 162 genes for which siRNA inhibition re- sulted in alteration in XBP1 splic- ing. These genes express diverse types of proteins modulating a wide range of cellular processes. Pathway analysis identified a set of genes implicated in the pathogenesis of breast cancer. Several genes, including BCL10, GCLM,andIGF1R, correlated with worse relapse-free survival (RFS) in an analysis of patients with triple-negative breast cancer (TNBC). However, in this cohort of 1,908 patients, only high GCLM expression correlated with worse RFS in both TNBC and non-TNBC patients. Altogether, our study revealed unidentified roles of novel pathwaysregulating the UPR, and these findings may serve as a paradigm for exploring novel therapeutic opportunities based on modulating the UPR.
    [Show full text]
  • Ire1α Regulates Macrophage Polarization, PD-L1 Expression And
    bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952457; this version posted February 17, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. PLOS Biology 1 IRE1a regulates macrophage polarization, PD-L1 expression and tumor survival 2 By 3 Alyssa Batista1*, Jeffrey J. Rodvold1*, Su Xian2, Stephen Searles1, Alyssa Lew1, Takao 4 Iwawaki3, Gonzalo Almanza1, T. Cameron Waller2, Jonathan Lin4, Kristen Jepsen5, Hannah 5 Carter2, and Maurizio Zanetti1 6 7 1 The Laboratory of Immunology 8 Department of Medicine and Moores Cancer Center 9 University of California, San Diego 10 9500 Gilman Drive 11 La Jolla, CA 92093-081 12 13 14 2Division of Medical Genetics; Department of Medicine, 15 And Bioinformatics and Systems Biology Program 16 University of California San Diego 17 La Jolla, CA 92093 18 19 3Laboratory for Cell Recovery Mechanisms 20 Brain Science Institute 21 RIKEN, 2-1 Hirosawa, Wako, Saitama 351-0198 22 Japan. 23 24 4Department of Pathology 25 Stanford University 26 300 Pasteur Drive 27 Palo Alto, CA 94305 28 29 5IGM Genomics Center 30 University of California, San Diego 31 9500 Gilman Drive 32 La Jolla, CA 92093-0612. 33 34 35 * These authors contributed equally 36 37 Corresponding author: Maurizio Zanetti 38 39 Ph: (858) 822-5412 40 FAX: (858) 822-5421 41 Email: [email protected] 42 43 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.02.17.952457; this version posted February 17, 2020.
    [Show full text]
  • Endoplasmic Reticulum Stress and the Protein Degradation System in Ophthalmic Diseases
    Endoplasmic reticulum stress and the protein degradation system in ophthalmic diseases Jing-Yao Song1, Xue-Guang Wang2, Zi-Yuan Zhang1, Lin Che1, Bin Fan1 and Guang-Yu Li1 1 Department of Ophthalmology, Second Hospital of Jilin University, ChangChun, China 2 Department of Traumatic Orthopedics, Third People's Hospital of Jinan, Jinan, China ABSTRACT Objective. Endoplasmic reticulum (ER) stress is involved in the pathogenesis of various ophthalmic diseases, and ER stress-mediated degradation systems play an important role in maintaining ER homeostasis during ER stress. The purpose of this review is to explore the potential relationship between them and to find their equilibrium sites. Design. This review illustrates the important role of reasonable regulation of the protein degradation system in ER stress-mediated ophthalmic diseases. There were 128 articles chosen for review in this study, and the keywords used for article research are ER stress, autophagy, UPS, ophthalmic disease, and ocular. Data sources. The data are from Web of Science, PubMed, with no language restrictions from inception until 2019 Jul. Results. The ubiquitin proteasome system (UPS) and autophagy are important degradation systems in ER stress. They can restore ER homeostasis, but if ER stress cannot be relieved in time, cell death may occur. However, they are not independent of each other, and the relationship between them is complementary. Therefore, we propose that ER stability can be achieved by adjusting the balance between them. Conclusion. The degradation system of ER stress, UPS and autophagy are interrelated. Because an imbalance between the UPS and autophagy can cause cell death, regulating that balance may suppress ER stress and protect cells against pathological stress damage.
    [Show full text]
  • Viral Mediated Tethering to SEL1L Facilitates ER-Associated Degradation of IRE1
    bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.330779; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 2 3 Viral mediated tethering to SEL1L facilitates ER-associated degradation of IRE1 4 5 6 Florian Hinte, Jendrik Müller, and Wolfram Brune* 7 8 Heinrich Pette Institute, Leibniz Institute for Experimental Virology, Hamburg, Germany 9 10 * corresponding author. [email protected] 11 12 Running head: MCMV M50 promotes ER-associated degradation of IRE1 13 14 15 Word count 16 Abstract: 249 17 Text: 3275 (w/o references) 18 Figures: 7 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.10.07.330779; this version posted October 9, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 19 Abstract 20 The unfolded protein response (UPR) and endoplasmic reticulum (ER)-associated 21 degradation (ERAD) are two essential components of the quality control system for proteins 22 in the secretory pathway. When unfolded proteins accumulate in the ER, UPR sensors such 23 as IRE1 induce the expression of ERAD genes, thereby increasing protein export from the ER 24 to the cytosol and subsequent degradation by the proteasome. Conversely, IRE1 itself is an 25 ERAD substrate, indicating that the UPR and ERAD regulate each other.
    [Show full text]
  • IRE1 (ERN1) Rabbit Polyclonal Antibody – TA336287 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for TA336287 IRE1 (ERN1) Rabbit Polyclonal Antibody Product data: Product Type: Primary Antibodies Applications: IF, WB Recommended Dilution: WB: 1:1000-1:2000, IF: 1:100 - 1:250 Reactivity: Human, Mouse, Rat (Does not react with: Primate) Host: Rabbit Clonality: Polyclonal Immunogen: A synthetic peptide within the human IRE1 alpha protein (within residues 700-800). [Swiss- Prot #O75460] Formulation: Tris-glycine, 150 mM NaCl, 0.05% Sodium Azide. Store at -20C. Avoid freeze-thaw cycles. Concentration: lot specific Purification: Immunogen affinity purified Conjugation: Unconjugated Storage: Store at -20°C as received. Stability: Stable for 12 months from date of receipt. Predicted Protein Size: 110 kDa Gene Name: endoplasmic reticulum to nucleus signaling 1 Database Link: NP_001424 Entrez Gene 78943 MouseEntrez Gene 498013 RatEntrez Gene 2081 Human O75460 This product is to be used for laboratory only. Not for diagnostic or therapeutic use. View online » ©2021 OriGene Technologies, Inc., 9620 Medical Center Drive, Ste 200, Rockville, MD 20850, US 1 / 3 IRE1 (ERN1) Rabbit Polyclonal Antibody – TA336287 Background: Unfolded protein response (UPR) signaling, mechanism used by eukaryotic cells to cope ER stress, is initiated by three ER-localized protein sensors: PERK (PKR-like ER kinase), ATF (activating transcription factor 6), and IRE1 alpha (inositol-requiring enzyme 1 alpha). UPR- responsive genes's transcriptional activation is regulated by ATF6 and IRE1-XBP1 pathways, and UPR serves three important functions: inhibition of protein translation to restore normal cell functions; activation of signaling to increase production of molecular chaperones involved in protein folding; and activation of signaling that leads to targeting of misfolded proteins in ER for ubiquitination and subsequent degradation; or when ER-stress is not relieved, UPR leads to apoptosis.
    [Show full text]
  • Hypoxic Regulation of EDN1, EDNRA, EDNRB, and ECE1 Gene Expressions in ERN1 Knockdown U87 Glioma Cells
    This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/ by-nc-nd/3.0), which permits copy and redistribute the material in any medium or format, provided the original work is properly cited. 250 ENDOCRINE REGULATIONS, Vol. 53, No. 4, 250–262, 2019 doi:10.2478/enr-2019-0025 Hypoxic regulation of EDN1, EDNRA, EDNRB, and ECE1 gene expressions in ERN1 knockdown U87 glioma cells Dmytro O. Minchenko1,2, Daria O. Tsymbal1, Olena O. Riabovol1, Yuliia M. Viletska1, Yuliia O. Lahanovska1, Myroslava Y. Sliusar1, Borys H. Bezrodnyi2, Oleksandr H. Minchenko1 1Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2National Bohomolets Medical University, Kyiv, Ukraine E-mail: [email protected] Objective. The aim of the present investigation was to study the effect of hypoxia on the expres- sion of genes encoding endothelin-1 (EDN1) and its cognate receptors (EDNRA and EDNRB) as well as endothelin converting enzyme 1 (ECE1) in U87 glioma cells in response to inhibition of en- doplasmic reticulum stress signaling mediated by ERN1/IRE1 (endoplasmic reticulum to nucleus signaling 1) for evaluation of their possible significance in the control of glioma growth through ERN1 and hypoxia. Methods. The expression level of EDN1, EDNRA, EDNRB, and ECE1 genes as well as micro- RNA miR-19, miR-96, and miR-206 was studied in control and ERN1 knockdown U87 glioma cells under hypoxia by quantitative polymerase chain reaction. Results. It was shown that the expression level of EDN1, EDNRA, EDNRB, and ECE1 genes was up-regulated in ERN1 knockdown glioma cells in comparison with the control glioma cells, be- ing more significant for endothelin-1.
    [Show full text]
  • ERN1 Knockdown Modifies the Effect of Glucose Deprivation on Homeobox Gene Expressions in U87 Glioma Cells
    This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/ licenses/ by-nc-nd/3.0), which permits copy and redistribute the material in any medium or format, provided the original work is properly cited. 196 ENDOCRINE REGULATIONS, Vol. 54, No. 3, 196–206, 2020 doi:10.2478/enr-2020-0022 ERN1 knockdown modifies the effect of glucose deprivation on homeobox gene expressions in U87 glioma cells Dariia O. Tsymbal1, Dmytro O. Minchenko1,2, Olena O. Khita1, Olha V. Rudnytska1, Yulia M. Viletska1, Yulia O. Lahanovska1, Qiuxia He3, Kechun Liu3, Oleksandr H. Minchenko1 1Department of Molecular Biology, Palladin Institute of Biochemistry, National Academy of Sciences of Ukraine, Kyiv, Ukraine; 2Department of Pediatrics, National Bohomolets Medical University, Kyiv, Ukraine; 3Biology Institute Shandong Academy of Sciences, Jinan, China E-mail: [email protected] Objective. The aim of the present investigation was to study the expression of genes encoding homeobox proteins ZEB2 (zinc finger E-box binding homeobox 2), TGIF1 (TGFB induced fac- tor homeobox 1), SPAG4 (sperm associated antigen 4), LHX1 (LIM homeobox 1), LHX2, LHX6, NKX3-1 (NK3 homeobox 1), and PRRX1 (paired related homeobox 1) in U87 glioma cells in response to glucose deprivation in control glioma cells and cells with knockdown of ERN1 (endo- plasmic reticulum to nucleus signaling 1), the major pathway of the endoplasmic reticulum stress signaling, for evaluation of it possible significance in the control of glioma growth through ERN1 signaling and chemoresistance. Methods. The expression level of homeobox family genes was studied in control (transfected by vector) and ERN1 knockdown U87 glioma cells under glucose deprivation condition by real-time quantitative polymerase chain reaction.
    [Show full text]
  • ERN1 and ALPK1 Inhibit Differentiation of Bi-Potential Tumor- Initiating Cells in Human Breast Cancer
    www.impactjournals.com/oncotarget/ Oncotarget, 2016, Vol. 7, (No. 50), pp: 83278-83293 Research Paper ERN1 and ALPK1 inhibit differentiation of bi-potential tumor- initiating cells in human breast cancer Juliane Strietz2,*, Stella S. Stepputtis2,3,*, Bogdan-Tiberius Preca2,3,*, Corinne Vannier2,3, Mihee M. Kim1, David J. Castro1, Qingyan Au1, Melanie Boerries3,5, Hauke Busch3,5, Pedro Aza-Blanc1, Susanne Heynen-Genel1, Peter Bronsert8,9,10, Bernhard Kuster6, Elmar Stickeler7, Thomas Brabletz4, Robert G. Oshima1,#, Jochen Maurer1,2,3,# 1Cancer Research Center, Sanford Burnham Prebys Medical Discovery Institute, La Jolla, CA, USA 2Department of Visceral Surgery, University Hospital Freiburg, German Cancer Consortium (DKTK), Freiburg, Germany 3German Cancer Research Center (DKFZ), Heidelberg, Germany 4Department of Experimental Medicine I, University of Erlangen-Nuernberg, Erlangen, Germany 5 Systems Biology of the Cellular Microenvironment at The DKFZ Partner Site Freiburg, German Cancer Consortium (DKTK), Institute of Molecular Medicine and Cell Research, Albert-Ludwigs-University Freiburg, Freiburg, Germany 6Technische Universitaet Muenchen, Partner Site of the German Cancer Consortium, Freising, Germany 7Department of OBGYN, University Clinic Aachen (UKA), Aachen, Germany 8Department of Surgical Pathology, University Medical Center Freiburg, Freiburg, Germany 9German Cancer Consortium (DKTK), German Cancer Research Center (DKFZ), Heidelberg, Germany 10Institute of Pathology, University Medical Center Freiburg, Freiburg, Germany *These authors have contributed equally and share first authorship #These authors have contributed equally and share last authorship Correspondence to: Jochen Maurer, email: [email protected] Robert G. Oshima, email: [email protected] Keywords:ALPK1, ERN1, differentiation therapy, human bi-potential tumor-initiating cells, kinase knockdown Received: March 21, 2016 Accepted: October 21, 2016 Published: November 04, 2016 ABSTRACT Cancers are heterogeneous by nature.
    [Show full text]
  • Detection of H3k4me3 Identifies Neurohiv Signatures, Genomic
    viruses Article Detection of H3K4me3 Identifies NeuroHIV Signatures, Genomic Effects of Methamphetamine and Addiction Pathways in Postmortem HIV+ Brain Specimens that Are Not Amenable to Transcriptome Analysis Liana Basova 1, Alexander Lindsey 1, Anne Marie McGovern 1, Ronald J. Ellis 2 and Maria Cecilia Garibaldi Marcondes 1,* 1 San Diego Biomedical Research Institute, San Diego, CA 92121, USA; [email protected] (L.B.); [email protected] (A.L.); [email protected] (A.M.M.) 2 Departments of Neurosciences and Psychiatry, University of California San Diego, San Diego, CA 92103, USA; [email protected] * Correspondence: [email protected] Abstract: Human postmortem specimens are extremely valuable resources for investigating trans- lational hypotheses. Tissue repositories collect clinically assessed specimens from people with and without HIV, including age, viral load, treatments, substance use patterns and cognitive functions. One challenge is the limited number of specimens suitable for transcriptional studies, mainly due to poor RNA quality resulting from long postmortem intervals. We hypothesized that epigenomic Citation: Basova, L.; Lindsey, A.; signatures would be more stable than RNA for assessing global changes associated with outcomes McGovern, A.M.; Ellis, R.J.; of interest. We found that H3K27Ac or RNA Polymerase (Pol) were not consistently detected by Marcondes, M.C.G. Detection of H3K4me3 Identifies NeuroHIV Chromatin Immunoprecipitation (ChIP), while the enhancer H3K4me3 histone modification was Signatures, Genomic Effects of abundant and stable up to the 72 h postmortem. We tested our ability to use H3K4me3 in human Methamphetamine and Addiction prefrontal cortex from HIV+ individuals meeting criteria for methamphetamine use disorder or not Pathways in Postmortem HIV+ Brain (Meth +/−) which exhibited poor RNA quality and were not suitable for transcriptional profiling.
    [Show full text]
  • Role of Endoplasmic Reticulum Stress Sensor Ire1α in Cellular Physiology, Calcium, ROS Signaling, and Metaflammation
    cells Review Role of Endoplasmic Reticulum Stress Sensor IRE1α in Cellular Physiology, Calcium, ROS Signaling, and Metaflammation Thoufiqul Alam Riaz 1 , Raghu Patil Junjappa 1 , Mallikarjun Handigund 2 , Jannatul Ferdous 3, Hyung-Ryong Kim 4,* and Han-Jung Chae 1,* 1 Department of Pharmacology, School of Medicine, Institute of New Drug Development, Jeonbuk National University, Jeonju 54907, Korea; toufi[email protected] (T.A.R.); [email protected] (R.P.J.) 2 Department of Laboratory Medicine, Jeonbuk National University, Medical School, Jeonju 54907, Korea; [email protected] 3 Department of Radiology and Research Institute of Clinical Medicine of Jeonbuk National University, Biomedical Research Institute of Jeonbuk National University Hospital, Jeonju 54907, Korea; [email protected] 4 College of Dentistry, Dankook University, Cheonan 31116, Korea * Correspondence: [email protected] (H.-R.K); [email protected] (H.-J.C) Received: 9 April 2020; Accepted: 6 May 2020; Published: 8 May 2020 Abstract: Inositol-requiring transmembrane kinase endoribonuclease-1α (IRE1α) is the most prominent and evolutionarily conserved unfolded protein response (UPR) signal transducer during endoplasmic reticulum functional upset (ER stress). A IRE1α signal pathway arbitrates yin and yang of cellular fate in objectionable conditions. It plays several roles in fundamental cellular physiology as well as in several pathological conditions such as diabetes, obesity, inflammation, cancer, neurodegeneration, and in many other diseases. Thus, further understanding of its molecular structure and mechanism of action during different cell insults helps in designing and developing better therapeutic strategies for the above-mentioned chronic diseases. In this review, recent insights into structure and mechanism of activation of IRE1α along with its complex regulating network were discussed in relation to their basic cellular physiological function.
    [Show full text]