DOCSLIB.ORG

Mouse Yipf4 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Yipf4 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Yipf4 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Yipf4 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Yipf4 gene (NCBI Reference Sequence: NM_026417 ; Ensembl: ENSMUSG00000024072 ) is located on Mouse chromosome 17. 6 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 6 (Transcript: ENSMUST00000024873). 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 Yipf4 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-14N10 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: Exon 2 starts from about 10.84% of the coding region. The knockout of Exon 2 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 2551 bp, and the size of intron 2 for 3'-loxP site insertion: 1422 bp. The size of effective cKO region: ~660 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 6 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Yipf4 Homology arm cKO region loxP site Page 2 of 7 https://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(7160bp) | A(26.48% 1896) | C(20.14% 1442) | T(31.06% 2224) | G(22.32% 1598) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. Significant high GC-content regions are found. It may be difficult to construct this targeting vector. Page 3 of 7 https://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% chr17 + 74489097 74492096 3000 browser details YourSeq 171 1651 1916 3000 92.5% chr1 + 74314104 74314491 388 browser details YourSeq 164 1468 1809 3000 87.0% chr7 - 42754839 42755273 435 browser details YourSeq 163 1651 2078 3000 87.8% chr7 - 27318253 27318588 336 browser details YourSeq 163 1651 2081 3000 86.4% chr11 + 68542045 68542317 273 browser details YourSeq 160 1626 1862 3000 94.0% chr11 + 57947079 57947371 293 browser details YourSeq 160 1269 1808 3000 87.6% chr1 + 93631943 93632372 430 browser details YourSeq 159 1266 1808 3000 85.8% chr10 - 21097226 21097486 261 browser details YourSeq 157 1639 1808 3000 94.0% chr4 + 155392126 155392291 166 browser details YourSeq 155 1639 1808 3000 96.5% chr16 - 17218936 17219600 665 browser details YourSeq 154 1647 1821 3000 97.0% chr6 - 140694705 140694884 180 browser details YourSeq 154 1651 1812 3000 97.6% chr13 - 64394051 64394212 162 browser details YourSeq 153 1640 1807 3000 95.9% chr6 - 96901430 96901602 173 browser details YourSeq 153 1639 1808 3000 94.1% chr16 - 48262150 48262317 168 browser details YourSeq 153 1649 1808 3000 98.2% chr2 + 103195317 103195487 171 browser details YourSeq 151 1642 1808 3000 95.9% chr7 + 123051779 123051956 178 browser details YourSeq 151 1639 1808 3000 95.3% chr6 + 148870711 148870892 182 browser details YourSeq 150 1471 1808 3000 92.7% chr9 - 114940090 114940429 340 browser details YourSeq 150 1651 1812 3000 96.3% chr9 + 64857929 64858090 162 browser details YourSeq 150 1651 1817 3000 93.0% chr14 + 7761637 7761792 156 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% chr17 + 74492757 74495756 3000 browser details YourSeq 443 452 1016 3000 93.8% chr6 + 44974256 44974966 711 browser details YourSeq 438 111 846 3000 90.9% chr2 + 5914967 5915596 630 browser details YourSeq 415 452 1016 3000 91.7% chr7 - 61705272 61705822 551 browser details YourSeq 398 250 846 3000 94.1% chr11 - 77256642 77257303 662 browser details YourSeq 384 451 970 3000 92.1% chr1 + 191308772 191309206 435 browser details YourSeq 383 452 958 3000 93.5% chr15 - 76775899 76776541 643 browser details YourSeq 376 394 860 3000 94.4% chr12 + 116730566 116731073 508 browser details YourSeq 370 451 970 3000 92.2% chr17 - 87033726 87034180 455 browser details YourSeq 369 452 970 3000 95.2% chr5 + 139219271 139219840 570 browser details YourSeq 366 445 851 3000 95.1% chr3 - 131008141 131008547 407 browser details YourSeq 366 451 856 3000 94.6% chr3 + 95548357 95548760 404 browser details YourSeq 363 454 846 3000 95.7% chr18 + 65912868 65913259 392 browser details YourSeq 362 444 846 3000 95.1% chr2 - 135248954 135249357 404 browser details YourSeq 362 452 859 3000 93.8% chr8 + 18534326 18534728 403 browser details YourSeq 361 394 846 3000 94.9% chr10 - 26951189 26951895 707 browser details YourSeq 361 449 852 3000 94.9% chrX + 67191505 67191909 405 browser details YourSeq 361 452 862 3000 95.0% chr9 + 94514197 94514606 410 browser details YourSeq 361 450 847 3000 95.5% chr2 + 44048304 44048702 399 browser details YourSeq 360 449 847 3000 95.3% chr3 - 149666249 149666648 400 Note: The 3000 bp section downstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Yipf4 Yip1 domain family, member 4 [ Mus musculus (house mouse) ] Gene ID: 67864, updated on 12-Aug-2019 Gene summary Official Symbol Yipf4 provided by MGI Official Full Name Yip1 domain family, member 4 provided by MGI Primary source MGI:MGI:1915114 See related Ensembl:ENSMUSG00000024072 Gene type protein coding RefSeq status VALIDATED Organism Mus musculus Lineage Eukaryota; Metazoa; Chordata; Craniata; Vertebrata; Euteleostomi; Mammalia; Eutheria; Euarchontoglires; Glires; Rodentia; Myomorpha; Muroidea; Muridae; Murinae; Mus; Mus Also known as 2310034L04Rik Expression Ubiquitous expression in adrenal adult (RPKM 43.1), duodenum adult (RPKM 26.4) and 28 other tissues See more Orthologs human all Genomic context Location: 17; 17 E2 See Yipf4 in Genome Data Viewer Exon count: 6 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (74489493..74500277) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (74888854..74899617) Chromosome 17 - NC_000083.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 6 transcripts Gene: Yipf4 ENSMUSG00000024072 Description Yip1 domain family, member 4 [Source:MGI Symbol;Acc:MGI:1915114] Gene Synonyms 2310034L04Rik Location Chromosome 17: 74,489,493-74,500,277 forward strand. GRCm38:CM001010.2 About this gene This gene has 6 transcripts (splice variants), 192 orthologues, 3 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Yipf4- ENSMUST00000024873.6 2133 246aa ENSMUSP00000024873.6 Protein coding CCDS28971 Q8C407 TSL:1 201 GENCODE basic APPRIS P1 Yipf4- ENSMUST00000234432.1 1991 39aa ENSMUSP00000157350.1 Nonsense mediated - A0A3Q4L376 - 202 decay Yipf4- ENSMUST00000234448.1 1082 No - lncRNA - - - 203 protein Yipf4- ENSMUST00000234939.1 481 No - lncRNA - - - 205 protein Yipf4- ENSMUST00000235064.1 343 No - lncRNA - - - 206 protein Yipf4- ENSMUST00000234853.1 166 No - lncRNA - - - 204 protein 30.79 kb Forward strand 74.48Mb 74.49Mb 74.50Mb 74.51Mb Genes (Comprehensive set... Yipf4-201 >protein coding Yipf4-205 >lncRNA Yipf4-204 >lncRNA Yipf4-202 >nonsense mediated decay Yipf4-203 >lncRNA Yipf4-206 >lncRNA Contigs < CT033749.8 Regulatory Build 74.48Mb 74.49Mb 74.50Mb 74.51Mb Reverse strand 30.79 kb Regulation Legend CTCF Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding processed transcript RNA gene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000024873 10.79 kb Forward strand Yipf4-201 >protein coding ENSMUSP00000024... Transmembrane heli... Low complexity (Seg) Pfam Yip1 domain PANTHER PTHR21236 PTHR21236:SF7 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 40 80 120 160 200 246 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
Load more

Recommended publications
  • RNA Editing at Baseline and Following Endoplasmic Reticulum Stress
    RNA Editing at Baseline and Following Endoplasmic Reticulum Stress By Allison Leigh Richards A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Human Genetics) in The University of Michigan 2015 Doctoral Committee: Professor Vivian G. Cheung, Chair Assistant Professor Santhi K. Ganesh Professor David Ginsburg Professor Daniel J. Klionsky Dedication To my father, mother, and Matt without whom I would never have made it ii Acknowledgements Thank you first and foremost to my dissertation mentor, Dr. Vivian Cheung. I have learned so much from you over the past several years including presentation skills such as never sighing and never saying “as you can see…” You have taught me how to think outside the box and how to create and explain my story to others. I would not be where I am today without your help and guidance. Thank you to the members of my dissertation committee (Drs. Santhi Ganesh, David Ginsburg and Daniel Klionsky) for all of your advice and support. I would also like to thank the entire Human Genetics Program, and especially JoAnn Sekiguchi and Karen Grahl, for welcoming me to the University of Michigan and making my transition so much easier. Thank you to Michael Boehnke and the Genome Science Training Program for supporting my work. A very special thank you to all of the members of the Cheung lab, past and present. Thank you to Xiaorong Wang for all of your help from the bench to advice on my career. Thank you to Zhengwei Zhu who has helped me immensely throughout my thesis even through my panic.
    [Show full text]
  • 17 - 19 October 2019
    THE 12TH INTERNATIONAL SYMPOSIUM ON HEALTH INFORMATICS AND BIOINFORMATICS T G T A A A T G A G A A G T T G G G T A A A A A A A A T T T A A A A A A G G A A A A A A A G G G G G G G G A A A G G G G G G T T G G G G G G G T T T T G G T T G G G T T T G G T A A T T G G T T T A A A A A A A A T T T A A A A A A T T A A A A A A A T A T T T T T T A A A T T T T G T A G A A T T T A A 17 - 19 OCTOBER 2019 HIBIT 2019 ABSTRACT BOOK THE 12TH INTERNATIONAL SYMPOSIUM ON HEALTH INFORMATICS AND BIOINFORMATICS TABLE OF CONTENTS 1 8 WELCOME MESSAGE KEYNOTE LECTURERS 2 19 ORGANIZING COMMITEE INVITED SPEAKERS 3 29 SCIENTIFIC COMMITEE SELECTED ABSTRACTS FOR ORAL PRESENTATIONS 6 61 PROGRAM POSTER PRESENTATIONS Welcome Message The International Symposium on Health Informatics and Bioinformatics, (HIBIT), now in its twelfth year HIBIT 2019, aims to bring together academics, researchers and practitioners who work in these popular and fulfilling areas and to create the much- needed synergy among medical, biological and information technology sectors. HIBIT is one of the few conferences emphasizing such synergy.
    [Show full text]
  • Human Induced Pluripotent Stem Cell–Derived Podocytes Mature Into Vascularized Glomeruli Upon Experimental Transplantation
    BASIC RESEARCH www.jasn.org Human Induced Pluripotent Stem Cell–Derived Podocytes Mature into Vascularized Glomeruli upon Experimental Transplantation † Sazia Sharmin,* Atsuhiro Taguchi,* Yusuke Kaku,* Yasuhiro Yoshimura,* Tomoko Ohmori,* ‡ † ‡ Tetsushi Sakuma, Masashi Mukoyama, Takashi Yamamoto, Hidetake Kurihara,§ and | Ryuichi Nishinakamura* *Department of Kidney Development, Institute of Molecular Embryology and Genetics, and †Department of Nephrology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan; ‡Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan; §Division of Anatomy, Juntendo University School of Medicine, Tokyo, Japan; and |Japan Science and Technology Agency, CREST, Kumamoto, Japan ABSTRACT Glomerular podocytes express proteins, such as nephrin, that constitute the slit diaphragm, thereby contributing to the filtration process in the kidney. Glomerular development has been analyzed mainly in mice, whereas analysis of human kidney development has been minimal because of limited access to embryonic kidneys. We previously reported the induction of three-dimensional primordial glomeruli from human induced pluripotent stem (iPS) cells. Here, using transcription activator–like effector nuclease-mediated homologous recombination, we generated human iPS cell lines that express green fluorescent protein (GFP) in the NPHS1 locus, which encodes nephrin, and we show that GFP expression facilitated accurate visualization of nephrin-positive podocyte formation in
    [Show full text]
  • Characterization of Five Transmembrane Proteins: with Focus on the Tweety, Sideroflexin, and YIP1 Domain Families
    fcell-09-708754 July 16, 2021 Time: 14:3 # 1 ORIGINAL RESEARCH published: 19 July 2021 doi: 10.3389/fcell.2021.708754 Characterization of Five Transmembrane Proteins: With Focus on the Tweety, Sideroflexin, and YIP1 Domain Families Misty M. Attwood1* and Helgi B. Schiöth1,2 1 Functional Pharmacology, Department of Neuroscience, Uppsala University, Uppsala, Sweden, 2 Institute for Translational Medicine and Biotechnology, Sechenov First Moscow State Medical University, Moscow, Russia Transmembrane proteins are involved in many essential cell processes such as signal transduction, transport, and protein trafficking, and hence many are implicated in different disease pathways. Further, as the structure and function of proteins are correlated, investigating a group of proteins with the same tertiary structure, i.e., the same number of transmembrane regions, may give understanding about their functional roles and potential as therapeutic targets. This analysis investigates the previously unstudied group of proteins with five transmembrane-spanning regions (5TM). More Edited by: Angela Wandinger-Ness, than half of the 58 proteins identified with the 5TM architecture belong to 12 families University of New Mexico, with two or more members. Interestingly, more than half the proteins in the dataset United States function in localization activities through movement or tethering of cell components and Reviewed by: more than one-third are involved in transport activities, particularly in the mitochondria. Nobuhiro Nakamura, Kyoto Sangyo University, Japan Surprisingly, no receptor activity was identified within this dataset in large contrast with Diego Bonatto, other TM groups. The three major 5TM families, which comprise nearly 30% of the Departamento de Biologia Molecular e Biotecnologia da UFRGS, Brazil dataset, include the tweety family, the sideroflexin family and the Yip1 domain (YIPF) Martha Martinez Grimes, family.
    [Show full text]
  • Transpochimeric Gene Trancripts (Tcgts) As Cancer Biomarkers
    (19) *EP003677693A1* (11) EP 3 677 693 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.: 08.07.2020 Bulletin 2020/28 C12Q 1/6886 (2018.01) (21) Application number: 19150151.9 (22) Date of filing: 03.01.2019 (84) Designated Contracting States: (72) Inventors: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB • TRONO, Didier GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO 1134 Vufflens-Chateau (CH) PL PT RO RS SE SI SK SM TR • SIMO RIUDALBAS, Laia Designated Extension States: 1004 Lausanne (CH) BA ME • PLANET LETSCHERT, Evarist Designated Validation States: 1113 ST-Saphorin-sur-Morges (CH) KH MA MD TN • DUC, Julien 1004 Lausanne (CH) (71) Applicants: • Ecole Polytechnique Federale De Lausanne (74) Representative: KATZAROV S.A. (EPFL) EPFL-TTO European Patent Attorneys 1015 Lausanne (CH) 12, Avenue des Morgines • Aarhus University 1213 Petit-Lancy (CH) 8000 Aarhus C (DK) (54) TRANSPOCHIMERIC GENE TRANCRIPTS (TCGTS) AS CANCER BIOMARKERS (57) The invention relates to methods for detecting or predicting the likelihood of a cancer in a subject as well as to compositions, pharmaceutical compositions and methods of treatment of cancer. EP 3 677 693 A1 Printed by Jouve, 75001 PARIS (FR) EP 3 677 693 A1 Description FIELD OF THE INVENTION 5 [0001] The invention relates to methods for detecting or predicting the likelihood of a cancer in a subject as well as to compositions, pharmaceutical compositions and methods of treatment of cancer. BACKGROUND OF THE INVENTION 10 [0002] Overall, current cancer drugs are inefficient in close to 75% of patients, and it is increasingly recognized that the future of cancer management lies in personalized approaches, whereby therapies are selected not just based on a tumor’s organ of origin but rather on a precise characterization of its molecular make-up and of the patient’s genetic background.
    [Show full text]
  • VU Research Portal
    VU Research Portal Genetic architecture and behavioral analysis of attention and impulsivity Loos, M. 2012 document version Publisher's PDF, also known as Version of record Link to publication in VU Research Portal citation for published version (APA) Loos, M. (2012). Genetic architecture and behavioral analysis of attention and impulsivity. General rights Copyright and moral rights for the publications made accessible in the public portal are retained by the authors and/or other copyright owners and it is a condition of accessing publications that users recognise and abide by the legal requirements associated with these rights. • Users may download and print one copy of any publication from the public portal for the purpose of private study or research. • You may not further distribute the material or use it for any profit-making activity or commercial gain • You may freely distribute the URL identifying the publication in the public portal ? Take down policy If you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediately and investigate your claim. E-mail address: [email protected] Download date: 28. Sep. 2021 Chapter 5 Independent genetic loci for sensorimotor gating and attentional performance in BXD recombinant inbred strains Maarten Loos, Jorn Staal, Tommy Pattij, Neuro-BSIK Mouse Phenomics consortium, August B. Smit, Sabine Spijker Genes Brain and Behavior, In Press 87 88 Sensorimotor gating and attention Abstract A startle reflex in response to an intense acoustic stimulus is inhibited when a barely detectable pulse precedes the startle stimulus by 30 – 500 ms.
    [Show full text]
  • YIPF2 Is a Novel Rab-GDF That Enhances HCC Malignant
    Qi et al. Cell Death and Disease (2019) 10:462 https://doi.org/10.1038/s41419-019-1709-8 Cell Death & Disease ARTICLE Open Access YIPF2 is a novel Rab-GDF that enhances HCC malignant phenotypes by facilitating CD147 endocytic recycle Shanshan Qi1, Linjia Su1,JingLi1, Pu Zhao2,QingZhang3, Xiuran Niu1, Jingyuan Liu1,GuheJia1,XiaoxuanWei1, Jan Tavernier4, Jianli Jiang5, Zhinan Chen5 and Sihe Zhang 1 Abstract An increased surface level of CIE (clathrin-independent endocytosis) proteins is a new feature of malignant neoplasms. CD147 is a CIE glycoprotein highly up-regulated in hepatocellular carcinoma (HCC). The ability to sort out the early endosome and directly target the recycling pathway confers on CD147 a prolonged surface half-life. However, current knowledge on CD147 trafficking to and from the cell-surface is limited. In this study, an MSP (membrane and secreted protein)-cDNA library was screened against EpoR/LR-F3/CD147EP-expressed cells by MAPPIT (mammalian protein–protein interaction trap). CD147 co-expressing with the new binder was investigated by GEPIA (gene expression profiling interactive analysis). The endocytosis, ER-Golgi trafficking and recycling of CD147 were measured by confocal imaging, flow cytometry, and biotin-labeled chase assays, respectively. Rab GTPase activation was checked by GST-RBD pull-down and MMP activity was measured by gelatin zymography. HCC malignant phenotypes were determined by cell adhesion, proliferation, migration, Transwell motility, and invasion assays. An ER-Golgi-resident transmembrane protein YIPF2 was identified as an intracellular binder to CD147. YIPF2 correlated and co-expressed fi 1234567890():,; 1234567890():,; 1234567890():,; 1234567890():,; with CD147, which is a survival predictor for HCC patients.
    [Show full text]
  • Modeling and Analysis of Acute Leukemia Using Human Hematopoietic Stem and Progenitor Cells
    Modeling and analysis of acute leukemia using human hematopoietic stem and progenitor cells A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirement for the degree of Doctor of Philosophy in the Molecular &Developmental Biology Graduate Program of the College of Medicine by Shan Lin BS, Tsinghua University, 2009 Dissertation Committee: James C. Mulloy, PhD (Chair) Geraldine Guasch, PhD Ashish R. Kumar, MD, PhD Daniel T. Starczynowski, PhD Yi Zheng, PhD Abstract For decades, elegant mouse models have yielded important insights into the complex biology of acute leukemia development. However, species differences between human and mouse could have significant influences on biological and translational applications. Therefore, human primary hematopoietic cells and xenograft mouse models have become important research tools in the field. In this report, we briefly review the methodologies that use human primary hematopoietic cells to model acute leukemia and examine the effects of leukemic oncogenes. The advantages and limitations of the human model system compared to syngeneic mouse models are discussed. The species-related complexity in human disease modeling is highlighted in the study establishing a faithful model of proB-ALL caused by MLL-AF4, the fusion product of the t(4;11). MLL-AF4 proB-ALL has poor prognosis, the lack of an accurate model hampers the study of disease pathobiology and therapeutic testing. We find human AF4 cDNA inhibits retroviral production and efficient transduction, this limitation can be overcome by fusing MLL with murine Af4, highly conserved with human AF4. Whereas MLL-Af4-transduced murine cells induce only AML, transduced human CD34+ cells produce proB-ALL faithful to t(4;11) disease, fully recapitulating the immunophenotypic and molecular aspects of the disease.
    [Show full text]
  • Seasonal Immunoregulation in a Naturally-Occurring Vertebrate
    Brown et al. BMC Genomics (2016) 17:369 DOI 10.1186/s12864-016-2701-7 RESEARCH ARTICLE Open Access Seasonal immunoregulation in a naturally- occurring vertebrate Martha Brown1, Pascal Hablützel1, Ida M. Friberg2, Anna G. Thomason2, Alexander Stewart3, Justin A. Pachebat1 and Joseph A. Jackson2* Abstract Background: Fishes show seasonal patterns of immunity, but such phenomena are imperfectly understood in vertebrates generally, even in humans and mice. As these seasonal patterns may link to infectious disease risk and individual condition, the nature of their control has real practical implications. Here we characterize seasonal dynamics in the expression of conserved vertebrate immunity genes in a naturally-occurring piscine model, the three-spined stickleback. Results: We made genome-wide measurements (RNAseq) of whole-fish mRNA pools (n = 36) at the end of summer and winter in contrasting habitats (riverine and lacustrine) and focussed on common trends to filter habitat-specific from overarching temporal responses. We corroborated this analysis with targeted year-round whole-fish gene expression (Q-PCR) studies in a different year (n = 478). We also considered seasonal tissue-specific expression (6 tissues) (n = 15) at a third contrasting (euryhaline) locality by Q-PCR, further validating the generality of the patterns seen in whole fish analyses. Extremes of season were the dominant predictor of immune expression (compared to sex, ontogeny or habitat). Signatures of adaptive immunity were elevated in late summer. In contrast, late winter was accompanied by signatures of innate immunity (including IL-1 signalling and non-classical complement activity) and modulated toll-like receptor signalling. Negative regulators of T-cell activity were prominent amongst winter-biased genes, suggesting that adaptive immunity is actively down-regulated during winter rather than passively tracking ambient temperature.
    [Show full text]
  • Neovascularization, Enhanced Inflammatory Response, and Age
    Biochemistry and Molecular Biology Neovascularization, Enhanced Inflammatory Response, and Age-Related Cone Dystrophy in the Nrl؊/؊Grk1؊/؊ Mouse Retina Rosanne M. Yetemian,1 Bruce M. Brown,1 and Cheryl M. Craft1,2 PURPOSE. The effects of aging and light exposure on cone cone photoreceptor homeostasis. (Invest Ophthalmol Vis Sci. photoreceptor survival were compared between mouse retinas 2010;51:6196–6206) DOI:10.1167/iovs.10-5452 of neural retina leucine zipper knockout (NrlϪ/Ϫ) mice and double-knockout mice lacking G-protein–coupled receptor ki- Ϫ Ϫ Ϫ Ϫ ignificant advances in bioinformatics have identified essen- nase 1 (Nrl / Grk1 / ). Stial genetic links and characterized basic molecular mecha- METHODS. Mice were reared in total darkness, ambient cyclic nisms driving the components of the visual G-protein–coupled light, or constant light, and their retinas were evaluated from 1 receptor (GPCR) signal transduction cascade leading to rod to 9 months of age using immunohistochemistry, electroreti- photoreceptor cell death. However, with a population of 3% to nography, and fluorescein angiography. Retinal gene expres- 5% cone photoreceptors in the mouse retina, the manifesta- sion and statistically significant probe sets were categorized tions of GPCR cascade disruption on cones have only recently using analysis software. Select gene expression changes were been studied with the help of the neural retina leucine zipper Ϫ Ϫ confirmed with quantitative RT-PCR. knockout (Nrl / ) mouse model.1 In humans, a loss-of-func- Ϫ/Ϫ Ϫ/Ϫ tion mutation in the NRL gene leads to an autosomal recessive RESULTS. In contrast to retinas from Nrl , those from Nrl Ϫ Ϫ disorder, enhanced S-cone syndrome, which causes an excess Grk1 / exhibit a progressive loss of the outer nuclear layer, number of S cones.
    [Show full text]
  • Early Vertebrate Whole Genome Duplications Were Predated by a Period of Intense Genome Rearrangement
    Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Early vertebrate whole genome duplications were predated by a period of intense genome rearrangement Andrew L. Hufton1, Detlef Groth1,2, Martin Vingron1, Hans Lehrach1, Albert J. Poustka1, Georgia Panopoulou1* 1. Max Planck for Molecular Genetics, Ihnestr. 73, 12169 Berlin, Germany. 2. Potsdam University, Bioinformatics Group, c/o Max Planck Institute of Molecular Plant Physiology, Am Muehlenberg 1, D-14476 Potsdam-Golm, Germany * Corresponding author: Max-Planck Institut für Molekulare Genetik, Ihnestrasse 73, D- 14195 Berlin Germany. email: [email protected], Tel: +49-30-84131235, Fax: +49- 30-84131128 Running title: Early vertebrate genome duplications and rearrangements Keywords: synteny, amphioxus, genome duplications, rearrangement rate, genome instability Downloaded from genome.cshlp.org on September 26, 2021 - Published by Cold Spring Harbor Laboratory Press Hufton et al. Abstract Researchers, supported by data from polyploid plants, have suggested that whole genome duplication (WGD) may induce genomic instability and rearrangement, an idea which could have important implications for vertebrate evolution. Benefiting from the newly released amphioxus genome sequence (Branchiostoma floridae), an invertebrate which researchers have hoped is representative of the ancestral chordate genome, we have used gene proximity conservation to estimate rates of genome rearrangement throughout vertebrates and some of their invertebrate ancestors. We find that, while amphioxus remains the best single source of invertebrate information about the early chordate genome, its genome structure is not particularly well conserved and it cannot be considered a fossilization of the vertebrate pre- duplication genome. In agreement with previous reports, we identify two WGD events in early vertebrates and another in teleost fish.
    [Show full text]
  • Towards Best-Practice Approaches for CRISPR/Cas9 Gene Engineering
    bioRxiv preprint doi: https://doi.org/10.1101/469544; this version posted December 2, 2018. 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-NC-ND 4.0 International license. Towards best-practice approaches for CRISPR/Cas9 gene engineering Claude Van Campenhout1,6, Pauline Cabochette2, Anne-Clémence Veillard1, Miklos Laczik1, Agnieszka Zelisko-Schmidt1, Céline Sabatel1, Maxime Dhainaut3, Benoit Vanhollebeke2,4 Cyril Gueydan5*, Véronique Kruys5* 1 Diagenode, SA, Liège Science Park, 4102 Seraing, Belgium 2 Laboratoire de Signalisation Neurovasculaire, 5 Laboratoire de Biologie Moléculaire du Gène, Faculté des Sciences, Université libre de Bruxelles (ULB), 12 rue des Profs. Jeener et Brachet, 6041 Gosselies, Belgium 3 Precision Immunology Institute, Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai, New York, NY 10029, USA 4 Walloon Excellence in Life Sciences and Biotechnology (WELBIO), Belgium 6 Present address: Laboratoire d’Anatomie Pathologique, Hôpital Erasme, Faculté de Médecine, Université Libre de Bruxelles (ULB), 808 route de Lennik, 1070 Bruxelles, Belgium *equally contributed Corresponding author : Véronique Kruys, [email protected] Keywords: gene targeting, quality control, methodology Abstract In recent years, CRISPR has evolved from “the curious sequence of unknown biological function” into a functional genome editing tool. The CRISPR/Cas9 technology is now delivering novel genetic models for fundamental research, drug screening, therapy development, rapid diagnostics and transcriptional modulation. Despite the apparent simplicity of the CRISPR/Cas9 system, the outcome of a genome editing experiment can be substantially impacted by technical parameters as well as biological considerations.
    [Show full text]