DOCSLIB.ORG

Mouse Stab2 Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Stab2 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Stab2 Knockout Project (CRISPR/Cas9) Objective: To create a Stab2 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stab2 gene (NCBI Reference Sequence: NM_138673 ; Ensembl: ENSMUSG00000035459 ) is located on Mouse chromosome 10. 69 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 69 (Transcript: ENSMUST00000035288). Exon 2~3 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for knock-out alleles exhibit no gross abnormaities. Mice homozygous for one null allele display elevated serum hyaluronic acid levels and decreased metastasis. Exon 2 starts from about 1.38% of the coding region. Exon 2~3 covers 3.26% of the coding region. The size of effective KO region: ~6291 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 69 Legends Exon of mouse Stab2 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 2 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(30.95% 619) | C(21.65% 433) | T(25.9% 518) | G(21.5% 430) Note: The 2000 bp section upstream of Exon 2 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. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(29.65% 593) | C(20.95% 419) | T(28.1% 562) | G(21.3% 426) Note: The 2000 bp section downstream of Exon 3 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 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr10 - 87003096 87005095 2000 browser details YourSeq 67 1433 1989 2000 70.7% chr14 + 58130802 58130936 135 browser details YourSeq 64 1425 1499 2000 86.8% chr2 + 158917817 158917884 68 browser details YourSeq 60 1428 1497 2000 89.9% chr4 - 56673840 56673908 69 browser details YourSeq 59 1428 1500 2000 92.7% chr9 + 96516798 96516872 75 browser details YourSeq 54 1426 1499 2000 82.6% chr17 + 16150099 16150165 67 browser details YourSeq 52 1432 1490 2000 86.8% chr9 - 117742893 117742945 53 browser details YourSeq 51 1436 1500 2000 96.3% chr18 - 19517318 19517385 68 browser details YourSeq 50 1433 1496 2000 96.4% chr3 - 109330045 109330120 76 browser details YourSeq 50 1426 1491 2000 82.8% chr17 - 43557079 43557140 62 browser details YourSeq 49 1433 1499 2000 94.6% chr2 + 24810775 24810865 91 browser details YourSeq 48 1435 1490 2000 86.8% chr2 - 62311239 62311291 53 browser details YourSeq 48 1428 1488 2000 81.5% chr17 - 70522653 70522706 54 browser details YourSeq 48 1435 1491 2000 84.4% chr11 + 109148491 109148541 51 browser details YourSeq 47 1426 1483 2000 94.4% chr15 - 78233861 78233919 59 browser details YourSeq 46 1428 1484 2000 94.2% chr4 - 58649513 58649576 64 browser details YourSeq 45 1453 1501 2000 97.9% chr12 - 101806221 101806283 63 browser details YourSeq 44 1432 1481 2000 94.0% chr16 - 72154178 72154227 50 browser details YourSeq 44 1436 1497 2000 79.3% chr14 + 114721951 114722006 56 browser details YourSeq 43 1433 1485 2000 95.7% chr15 - 17625914 17625966 53 Note: The 2000 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 2000 1 2000 2000 100.0% chr10 - 86994805 86996804 2000 browser details YourSeq 138 638 1034 2000 90.2% chr10 + 10974431 11002243 27813 browser details YourSeq 123 469 777 2000 93.7% chr7 - 44887985 44888480 496 browser details YourSeq 122 549 805 2000 83.0% chr1 - 21522655 21522852 198 browser details YourSeq 115 627 1245 2000 91.3% chr11 - 57679840 57804872 125033 browser details YourSeq 110 638 799 2000 84.7% chrX - 94406237 94406390 154 browser details YourSeq 110 638 799 2000 84.2% chr11 + 46643745 46643896 152 browser details YourSeq 110 638 777 2000 89.1% chr1 + 192910497 192910635 139 browser details YourSeq 108 638 779 2000 88.3% chr7 - 112860705 112860842 138 browser details YourSeq 107 638 779 2000 86.4% chr11 - 3311564 3311701 138 browser details YourSeq 106 638 778 2000 86.9% chr13 - 65362445 65362581 137 browser details YourSeq 106 638 791 2000 88.5% chr10 - 58558139 58558301 163 browser details YourSeq 106 638 778 2000 89.0% chr1 - 132396193 132396336 144 browser details YourSeq 106 638 779 2000 87.9% chr11 + 86678463 86678602 140 browser details YourSeq 106 647 1035 2000 86.4% chr10 + 3738506 3738914 409 browser details YourSeq 105 638 776 2000 88.0% chr13 - 106831456 106831590 135 browser details YourSeq 105 638 777 2000 87.5% chr11 - 50081239 50081375 137 browser details YourSeq 105 638 798 2000 85.0% chr1 - 93538902 93539046 145 browser details YourSeq 104 633 779 2000 86.1% chr11 - 5696400 5696544 145 browser details YourSeq 104 638 778 2000 91.4% chr5 + 139116744 139116885 142 Note: The 2000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Stab2 stabilin 2 [ Mus musculus (house mouse) ] Gene ID: 192188, updated on 12-Aug-2019 Gene summary Official Symbol Stab2 provided by MGI Official Full Name stabilin 2 provided by MGI Primary source MGI:MGI:2178743 See related Ensembl:ENSMUSG00000035459 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 FELL; FEEL-2; STAB-2; MFEEL-2 Expression Biased expression in spleen adult (RPKM 16.0), liver E18 (RPKM 6.2) and 8 other tissues See more Orthologs human all Genomic context Location: 10; 10 C1 See Stab2 in Genome Data Viewer Exon count: 74 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 10 NC_000076.6 (86841194..87008038, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 10 NC_000076.5 (86303955..86470687, complement) Chromosome 10 - NC_000076.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 7 transcripts Gene: Stab2 ENSMUSG00000035459 Description stabilin 2 [Source:MGI Symbol;Acc:MGI:2178743] Gene Synonyms FEEL-2, STAB-2 Location Chromosome 10: 86,841,198-87,008,025 reverse strand. GRCm38:CM001003.2 About this gene This gene has 7 transcripts (splice variants), 226 orthologues, 2 paralogues, is a member of 1 Ensembl protein family and is associated with 4 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Stab2- ENSMUST00000035288.16 8228 2559aa ENSMUSP00000048309.8 Protein coding CCDS36021 E5RKF9 TSL:1 201 Q8R4U0 GENCODE basic APPRIS P1 Stab2- ENSMUST00000219341.2 3502 870aa ENSMUSP00000151465.2 Nonsense mediated - A0A1W2P6Y4 CDS 5' 205 decay incomplete TSL:5 Stab2- ENSMUST00000219612.1 502 No - Retained intron - - TSL:3 206 protein Stab2- ENSMUST00000219659.1 442 No - Retained intron - - TSL:5 207 protein Stab2- ENSMUST00000218408.1 1237 No - lncRNA - - TSL:1 203 protein Stab2- ENSMUST00000218366.1 720 No - lncRNA - - TSL:1 202 protein Stab2- ENSMUST00000219280.1 523 No - lncRNA - - TSL:3 204 protein Page 7 of 9 https://www.alphaknockout.com 186.83 kb Forward strand 86.85Mb 86.90Mb 86.95Mb 87.00Mb Genes Nt5dc3-201 >protein coding Gm16280-201 >lncRNA Gm16271-201 >lncRNA (Comprehensive set... Nt5dc3-202 >retained intron Gm16270-201 >transcribed processed pseudogene Gm16269-201 >processed pseudogene Gm49358-201 >protein coding Gm16268-201 >lncRNA Contigs < AC112790.5 < AC025501.19 Genes (Comprehensive set... < Stab2-201protein coding < Stab2-205nonsense mediated decay < Stab2-203lncRNA < Stab2-204lncRNA < Stab2-206retained intron < Stab2-202lncRNA < Stab2-207retained intron Regulatory Build 86.85Mb 86.90Mb 86.95Mb 87.00Mb Reverse strand 186.83 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Transcription Factor Binding Site Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene processed transcript RNA gene Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000035288 < Stab2-201protein coding Reverse strand 166.83 kb ENSMUSP00000048... Transmembrane heli... MobiDB lite Low complexity (Seg) Cleavage site (Sign... Superfamily SSF57196 C-type lectin fold FAS1 domain superfamily SMART EGF-like domain Link domain EGF-like calcium-binding domain FAS1 domain Laminin EGF domain Pfam FAS1 domain Link domain EGF domain PROSITE profiles FAS1 domain EGF-like domain Link domain PROSITE patterns EGF-like, conserved site Link domain EGF-like, conserved site PANTHER PTHR24038 PTHR24038:SF0 Gene3D 2.170.300.10 C-type lectin-like/link domain superfamily FAS1 domain superfamily 2.10.25.10 CDD cd00054 cd00055 All sequence SNPs/i..
Load more

Recommended publications
  • Supplementary Table 1: Adhesion Genes Data Set
    Supplementary Table 1: Adhesion genes data set PROBE Entrez Gene ID Celera Gene ID Gene_Symbol Gene_Name 160832 1 hCG201364.3 A1BG alpha-1-B glycoprotein 223658 1 hCG201364.3 A1BG alpha-1-B glycoprotein 212988 102 hCG40040.3 ADAM10 ADAM metallopeptidase domain 10 133411 4185 hCG28232.2 ADAM11 ADAM metallopeptidase domain 11 110695 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 195222 8038 hCG40937.4 ADAM12 ADAM metallopeptidase domain 12 (meltrin alpha) 165344 8751 hCG20021.3 ADAM15 ADAM metallopeptidase domain 15 (metargidin) 189065 6868 null ADAM17 ADAM metallopeptidase domain 17 (tumor necrosis factor, alpha, converting enzyme) 108119 8728 hCG15398.4 ADAM19 ADAM metallopeptidase domain 19 (meltrin beta) 117763 8748 hCG20675.3 ADAM20 ADAM metallopeptidase domain 20 126448 8747 hCG1785634.2 ADAM21 ADAM metallopeptidase domain 21 208981 8747 hCG1785634.2|hCG2042897 ADAM21 ADAM metallopeptidase domain 21 180903 53616 hCG17212.4 ADAM22 ADAM metallopeptidase domain 22 177272 8745 hCG1811623.1 ADAM23 ADAM metallopeptidase domain 23 102384 10863 hCG1818505.1 ADAM28 ADAM metallopeptidase domain 28 119968 11086 hCG1786734.2 ADAM29 ADAM metallopeptidase domain 29 205542 11085 hCG1997196.1 ADAM30 ADAM metallopeptidase domain 30 148417 80332 hCG39255.4 ADAM33 ADAM metallopeptidase domain 33 140492 8756 hCG1789002.2 ADAM7 ADAM metallopeptidase domain 7 122603 101 hCG1816947.1 ADAM8 ADAM metallopeptidase domain 8 183965 8754 hCG1996391 ADAM9 ADAM metallopeptidase domain 9 (meltrin gamma) 129974 27299 hCG15447.3 ADAMDEC1 ADAM-like,
    [Show full text]
  • Epigenetic Regulation of Murine Stab2 Expression in Determining Atherosclerotic Susceptibility
    EPIGENETIC REGULATION OF MURINE STAB2 EXPRESSION IN DETERMINING ATHEROSCLEROTIC SUSCEPTIBILITY Sharlene Dong A thesis submitted to the faculty of the University of North Carolina at Chapel Hill in partial fulfillment of the requirements for the degree of Master of Science in the Department of Nutrition (Nutritional Biochemistry) in the Gillings School of Global Public Health Chapel Hill 2017 Approved by: Nobuyo Maeda Rosalind Coleman Eric L. Klett i © 2017 Sharlene Dong ALL RIGHTS RESERVED ii ABSTRACT Sharlene Dong: Epigenetic regulation of murine Stab2 expression in determining atherosclerotic susceptibility (Under the direction of Nobuyo Maeda) Atherosclerosis is a progressive disease characterized by the accumulation of lipids and fibrous elements in the arteries. In mice, the aortic arch and root show distinct patterns of lesion development. A quantitative trait locus (QTL) analysis of an intercross between apoE-null mice on 129S6 and DBA/2J backgrounds has revealed a significant locus on chromosome 10, Aath5, that affects plaque size in the arch. One candidate gene in the Aath5 locus is the Stab2 gene, which encodes a receptor for hyaluronan. The DBA allele of the Stab2 gene includes a unique intracisternal A-particle (IAP) retrotransposon inserted in reverse in the promoter region, which drives Stab2 expression. Methylation status of this region was analyzed through allele-specific bisulfite sequencing and correlated with ectopic Stab2 expression in the heart and kidneys. Thus, epigenetic regulation of this IAP element may play a role in regulating Stab2 expression and affect atherosclerotic susceptibility in the aortic arch. iii To my mom and dad and all my friends Sincerely thank you for your unwavering support in me.
    [Show full text]
  • Prognostic Values of Stabilin-2 in Hepatocellular Carcinoma
    Prognostic Values of Stabilin-2 in Hepatocellular Carcinoma Zhicheng Du Zhengzhou University First Aliated Hospital https://orcid.org/0000-0002-1257-6619 Pengfei Zhu Zhengzhou University First Aliated Hospital Long Yu Zhengzhou University First Aliated Hospital Kunlun Chen Zhengzhou University First Aliated Hospital Janwen Ye Zhengzhou University First Aliated Hospital Wenlong Zhai ( [email protected] ) https://orcid.org/0000-0003-2559-2105 Primary research Keywords: Hepatocellular carcinoma, Stabilin-2, biomarker, prognostic Posted Date: May 11th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-27280/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/20 Abstract Background: Hepatocellular carcinoma (HCC) is the primary malignancy of the liver. However, biomarkers for early HCC diagnosis are not available. Stabilin (STAB) proteins are scavenger receptors involved in apoptosis and clearance of hyaluronic acid .The role of STAB in HCC has not been previously explored; therefore, the aim of this study was to assess whether STAB gene expression can be used as a novel HCC biomarker. Materials and Methods: Data on 370 HCC patients in the Cancer Genome Atlas database and 221 patients in the Gene Expression Comprehensive Database were retrieved and analyzed. Kaplan–Meier analysis and Cox regression model were used to calculate median survival time using hazard ratio (HR) and 95% condence interval (CI). Results: The Gene Expression Omnibus dataset showed that high Stabilin-2STAB2 expression implies longer overall survival (HR after correction = 0.541; 95% CI, 0.339–0.865; p = 0.0182, after correction p = 0.010) and longer recurrence-free survival time (adjusted HR = 0.554; 95% CI, 0.376-0.816; p = 0.0085, adjusted p = 0.003).
    [Show full text]
  • Transcriptional Landscape of Pulmonary Lymphatic Endothelial Cells During Fetal Gestation
    RESEARCH ARTICLE Transcriptional landscape of pulmonary lymphatic endothelial cells during fetal gestation 1,2 3 1 1,4 Timothy A. Norman, Jr.ID *, Adam C. Gower , Felicia Chen , Alan Fine 1 Pulmonary Center, Boston University School of Medicine, Boston, Massachusetts, United States of America, 2 Pathology & Laboratory Medicine, Boston University School of Medicine, Boston, Massachusetts, United States of America, 3 Clinical and Translational Science Institute, Boston University School of Medicine, Boston, Massachusetts, United States of America, 4 Boston Veteran's Hospital, West Roxbury, a1111111111 Massachusetts, United States of America a1111111111 a1111111111 * [email protected] a1111111111 a1111111111 Abstract The genetic programs responsible for pulmonary lymphatic maturation prior to birth are not known. To address this gap in knowledge, we developed a novel cell sorting strategy to col- OPEN ACCESS lect fetal pulmonary lymphatic endothelial cells (PLECs) for global transcriptional profiling. Citation: Norman TA, Jr., Gower AC, Chen F, Fine A We identified PLECs based on their unique cell surface immunophenotype (CD31+/Vegfr3 (2019) Transcriptional landscape of pulmonary lymphatic endothelial cells during fetal gestation. +/Lyve1+/Pdpn+) and isolated them from murine lungs during late gestation (E16.5, E17.5, PLoS ONE 14(5): e0216795. https://doi.org/ E18.5). Gene expression profiling was performed using whole-genome microarrays, and 10.1371/journal.pone.0216795 1,281 genes were significantly differentially expressed with respect to time (FDR q < 0.05) Editor: Vladimir V. Kalinichenko, Cincinnati and grouped into six clusters. Two clusters containing a total of 493 genes strongly upregu- Children's Hospital Medical Center, UNITED lated at E18.5 were significantly enriched in genes with functional annotations correspond- STATES ing to innate immune response, positive regulation of angiogenesis, complement & Received: October 19, 2018 coagulation cascade, ECM/cell-adhesion, and lipid metabolism.
    [Show full text]
  • Comprehensive Analysis Reveals Novel Gene Signature in Head and Neck Squamous Cell Carcinoma: Predicting Is Associated with Poor Prognosis in Patients
    5892 Original Article Comprehensive analysis reveals novel gene signature in head and neck squamous cell carcinoma: predicting is associated with poor prognosis in patients Yixin Sun1,2#, Quan Zhang1,2#, Lanlin Yao2#, Shuai Wang3, Zhiming Zhang1,2 1Department of Breast Surgery, The First Affiliated Hospital of Xiamen University, School of Medicine, Xiamen University, Xiamen, China; 2School of Medicine, Xiamen University, Xiamen, China; 3State Key Laboratory of Cellular Stress Biology, School of Life Sciences, Xiamen University, Xiamen, China Contributions: (I) Conception and design: Y Sun, Q Zhang; (II) Administrative support: Z Zhang; (III) Provision of study materials or patients: Y Sun, Q Zhang; (IV) Collection and assembly of data: Y Sun, L Yao; (V) Data analysis and interpretation: Y Sun, S Wang; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. #These authors contributed equally to this work. Correspondence to: Zhiming Zhang. Department of Surgery, The First Affiliated Hospital of Xiamen University, Xiamen, China. Email: [email protected]. Background: Head and neck squamous cell carcinoma (HNSC) remains an important public health problem, with classic risk factors being smoking and excessive alcohol consumption and usually has a poor prognosis. Therefore, it is important to explore the underlying mechanisms of tumorigenesis and screen the genes and pathways identified from such studies and their role in pathogenesis. The purpose of this study was to identify genes or signal pathways associated with the development of HNSC. Methods: In this study, we downloaded gene expression profiles of GSE53819 from the Gene Expression Omnibus (GEO) database, including 18 HNSC tissues and 18 normal tissues.
    [Show full text]
  • Genes Associated with Anhedonia
    Ren et al. Translational Psychiatry (2018) 8:150 DOI 10.1038/s41398-018-0198-3 Translational Psychiatry ARTICLE Open Access Genes associated with anhedonia: a new analysis in a large clinical trial (GENDEP) Hongyan Ren1, Chiara Fabbri2, Rudolf Uher3, Marcella Rietschel 4,OleMors5, Neven Henigsberg 6,JoannaHauser7, Astrid Zobel8, Wolfgang Maier8, Mojca Z. Dernovsek9,DanielSouery10, Annamaria Cattaneo11, Gerome Breen2, Ian W. Craig2,AnneE.Farmer2,PeterMcGuffin2, Cathryn M. Lewis 2 and Katherine J. Aitchison 1,2 Abstract A key feature of major depressive disorder (MDD) is anhedonia, which is a predictor of response to antidepressant treatment. In order to shed light on its genetic underpinnings, we conducted a genome-wide association study (GWAS) followed by investigation of biological pathway enrichment using an anhedonia dimension for 759 patients with MDD in the GENDEP study. The GWAS identified 18 SNPs associated at genome-wide significance with the top one being an intronic SNP (rs9392549) in PRPF4B (pre-mRNA processing factor 4B) located on chromosome 6 (P = 2.07 × 10−9) while gene-set enrichment analysis returned one gene ontology term, axon cargo transport (GO: 0008088) with a nominally significant P value (1.15 × 10−5). Furthermore, our exploratory analysis yielded some interesting, albeit not statistically significant genetic correlation with Parkinson’s Disease and nucleus accumbens gray matter. In addition, polygenic risk scores (PRSs) generated from our association analysis were found to be able to predict treatment efficacy of the antidepressants in this study. In conclusion, we found some markers significantly associated with anhedonia, and some suggestive findings of related pathways and biological functions, which could be further investigated in other studies.
    [Show full text]
  • Quantitative Trait Loci Mapping of Macrophage Atherogenic Phenotypes
    QUANTITATIVE TRAIT LOCI MAPPING OF MACROPHAGE ATHEROGENIC PHENOTYPES BRIAN RITCHEY Bachelor of Science Biochemistry John Carroll University May 2009 submitted in partial fulfillment of requirements for the degree DOCTOR OF PHILOSOPHY IN CLINICAL AND BIOANALYTICAL CHEMISTRY at the CLEVELAND STATE UNIVERSITY December 2017 We hereby approve this thesis/dissertation for Brian Ritchey Candidate for the Doctor of Philosophy in Clinical-Bioanalytical Chemistry degree for the Department of Chemistry and the CLEVELAND STATE UNIVERSITY College of Graduate Studies by ______________________________ Date: _________ Dissertation Chairperson, Johnathan D. Smith, PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, David J. Anderson, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Baochuan Guo, PhD Department of Chemistry, Cleveland State University ______________________________ Date: _________ Dissertation Committee member, Stanley L. Hazen, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Renliang Zhang, MD PhD Department of Cellular and Molecular Medicine, Cleveland Clinic ______________________________ Date: _________ Dissertation Committee member, Aimin Zhou, PhD Department of Chemistry, Cleveland State University Date of Defense: October 23, 2017 DEDICATION I dedicate this work to my entire family. In particular, my brother Greg Ritchey, and most especially my father Dr. Michael Ritchey, without whose support none of this work would be possible. I am forever grateful to you for your devotion to me and our family. You are an eternal inspiration that will fuel me for the remainder of my life. I am extraordinarily lucky to have grown up in the family I did, which I will never forget.
    [Show full text]
  • Ectopic Expression of the Stabilin2 Gene Triggered by an Intracisternal a Particle (IAP) Element in DBA/2J Strain of Mice
    Mammalian Genome (2020) 31:2–16 https://doi.org/10.1007/s00335-019-09824-1 Ectopic expression of the Stabilin2 gene triggered by an intracisternal A particle (IAP) element in DBA/2J strain of mice Nobuyo Maeda‑Smithies1 · Sylvia Hiller1 · Sharlene Dong1,3 · Hyung‑Suk Kim1 · Brian J. Bennett2,4 · Yukako Kayashima1 Received: 1 November 2019 / Accepted: 29 December 2019 / Published online: 7 January 2020 © The Author(s) 2020 Abstract Stabilin2 (Stab2) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the Stab2 locus. Stab2 mRNA in the LSECs was signifcantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), DBA in the promoter region of Stab2 which likely interferes with normal expression in the LSECs. In contrast, in other tissues DBA of DBA/2J mice, the IAP drives high ectopic Stab2 transcription starting within the 5′ long terminal repeat of IAP in a DBA reverse orientation and continuing through the downstream Stab2 . Ectopic transcription requires the Stab2-IAP element but is dominantly suppressed by the presence of loci on 59.7–73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9–73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-fnger proteins that target transposable elements-derived sequences and DBA repress their expression.
    [Show full text]
  • Differential Gene Expression by RNA-Seq Analysis of the Primo Vessel in the Rabbit Lymph
    J Acupunct Meridian Stud 2019;12(1):11e19 Available online at www.sciencedirect.com Journal of Acupuncture and Meridian Studies journal homepage: www.jams-kpi.com Research Article Differential Gene Expression by RNA-Seq Analysis of the Primo Vessel in the Rabbit Lymph Jun-Young Shin, Sang-Heon Choi, Da-Woon Choi, Ye-Jin An, Jae-Hyuk Seo, Jong-Gu Choi, Min-Suk Rho, Sang-Suk Lee* Department of Oriental Biomedical Engineering, Sangji University, Wonju, Republic of Korea Available online 28 October 2018 Received: May 31, 2018 Abstract Revised: Jul 26, 2018 For the connectome of primo vascular system, some long-type primo vessels dyed with Accepted: Oct 23, 2018 Alcian blue injected into inguinal nodes, abdominal node, and axially nodes were visual- ized, which passed over around the vena cava of the rabbit. The Alcian blue dye revealed KEYWORDS primo vessels and colored blue in the rabbit lymph vessels. The length of long-type primo e m gene expression level; vessels was 18 cm on average, of which diameters were about 20 30 m, and the lymph e m lymph node; vessels had diameters of 100 150 m. Three different tissues of pure primo vessel, mixed primo connectome; primo þ lymph vessel, and only lymph vessel were made to undergo RNA-Seq analysis by RNA-Seq analysis next-generation sequencing. We also analyzed differentially expressed genes (DEGs) from the RNA-Seq data, in which 30 genes of the primo vessels, primo þ lymph vessels, and lymph vessels were selected for primo marker candidates. From the plot of DEG analysis, 10 genes had remarkably different expression pattern on the Group 1 (primo vessel) vs Group 3 (lymph vessel).
    [Show full text]
  • Mouse Stab2 Conditional Knockout Project (CRISPR/Cas9)
    https://www.alphaknockout.com Mouse Stab2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Stab2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Stab2 gene (NCBI Reference Sequence: NM_138673 ; Ensembl: ENSMUSG00000035459 ) is located on Mouse chromosome 10. 69 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 69 (Transcript: ENSMUST00000035288). Exon 4 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Stab2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-233D10 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 knock-out alleles exhibit no gross abnormaities. Mice homozygous for one null allele display elevated serum hyaluronic acid levels and decreased metastasis. Exon 4 starts from about 4.64% of the coding region. The knockout of Exon 4 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 15628 bp, and the size of intron 4 for 3'-loxP site insertion: 1090 bp. The size of effective cKO region: ~586 bp. The cKO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 4 5 69 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Stab2 Homology arm cKO region loxP site Page 2 of 8 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.
    [Show full text]
  • An Integrative Genomic Analysis of the Longshanks Selection Experiment for Longer Limbs in Mice
    bioRxiv preprint doi: https://doi.org/10.1101/378711; this version posted August 19, 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. 1 Title: 2 An integrative genomic analysis of the Longshanks selection experiment for longer limbs in mice 3 Short Title: 4 Genomic response to selection for longer limbs 5 One-sentence summary: 6 Genome sequencing of mice selected for longer limbs reveals that rapid selection response is 7 due to both discrete loci and polygenic adaptation 8 Authors: 9 João P. L. Castro 1,*, Michelle N. Yancoskie 1,*, Marta Marchini 2, Stefanie Belohlavy 3, Marek 10 Kučka 1, William H. Beluch 1, Ronald Naumann 4, Isabella Skuplik 2, John Cobb 2, Nick H. 11 Barton 3, Campbell Rolian2,†, Yingguang Frank Chan 1,† 12 Affiliations: 13 1. Friedrich Miescher Laboratory of the Max Planck Society, Tübingen, Germany 14 2. University of Calgary, Calgary AB, Canada 15 3. IST Austria, Klosterneuburg, Austria 16 4. Max Planck Institute for Cell Biology and Genetics, Dresden, Germany 17 Corresponding author: 18 Campbell Rolian 19 Yingguang Frank Chan 20 * indicates equal contribution 21 † indicates equal contribution 22 Abstract: 23 Evolutionary studies are often limited by missing data that are critical to understanding the 24 history of selection. Selection experiments, which reproduce rapid evolution under controlled 25 conditions, are excellent tools to study how genomes evolve under strong selection. Here we 1 bioRxiv preprint doi: https://doi.org/10.1101/378711; this version posted August 19, 2018.
    [Show full text]
  • INTRODUCING a NOVEL METHOD for GENETIC ANALYSIS of AUTISM SPECTRUM DISORDER Sepideh Nouri
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 12-2013 INTRODUCING A NOVEL METHOD FOR GENETIC ANALYSIS OF AUTISM SPECTRUM DISORDER sepideh nouri Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Bioinformatics Commons, Computational Biology Commons, and the Genomics Commons Recommended Citation nouri, sepideh, "INTRODUCING A NOVEL METHOD FOR GENETIC ANALYSIS OF AUTISM SPECTRUM DISORDER" (2013). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 417. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/417 This Thesis (MS) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. INTRODUCING A NOVEL METHOD FOR GENETIC ANALYSIS OF AUTISM SPECTRUM DISORDER by Sepideh Nouri, M.S. APPROVED: Eric Boerwinkle, Ph.D., Supervisor Kim-Anh Do, Ph.D. Alanna Morrison, Ph.D. James Hixson, Ph.D. Paul Scheet, Ph.D. APPROVED: Dean, The University of Texas Graduate School of Biomedical Sciences at Houston INTRODUCING A NOVEL METHOD FOR GENETIC ANALYSIS OF AUTISM SPECTRUM DISORDER A THESIS Presented to the Faculty of The University of Texas Health Science Center at Houston and The University of Texas M.
    [Show full text]