Mouse Dhx32 Knockout Project (CRISPR/Cas9)

Total Page:16

File Type:pdf, Size:1020Kb

Mouse Dhx32 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Dhx32 Knockout Project (CRISPR/Cas9) Objective: To create a Dhx32 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Dhx32 gene (NCBI Reference Sequence: NM_133941 ; Ensembl: ENSMUSG00000030986 ) is located on Mouse chromosome 7. 12 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000033290). Exon 4~7 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: Exon 4 starts from about 22.1% of the coding region. Exon 4~7 covers 38.97% of the coding region. The size of effective KO region: ~8834 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 4 5 6 7 12 Legends Exon of mouse Dhx32 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 4 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section downstream of Exon 7 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(28.1% 562) | C(20.95% 419) | T(27.4% 548) | G(23.55% 471) Note: The 2000 bp section upstream of Exon 4 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(22.6% 452) | C(23.2% 464) | T(28.3% 566) | G(25.9% 518) Note: The 2000 bp section downstream of Exon 7 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% chr7 - 133742802 133744801 2000 browser details YourSeq 59 455 574 2000 90.5% chr3 + 131478388 131478528 141 browser details YourSeq 56 515 592 2000 82.9% chr11 - 86228625 86228698 74 browser details YourSeq 53 501 584 2000 93.5% chr7 - 80752410 80822082 69673 browser details YourSeq 52 529 589 2000 93.5% chr11 - 98006769 98006830 62 browser details YourSeq 52 472 575 2000 85.2% chr2 + 161104170 161104296 127 browser details YourSeq 51 501 573 2000 87.0% chr19 + 53691865 53691945 81 browser details YourSeq 50 520 592 2000 88.1% chr9 - 67576247 67576319 73 browser details YourSeq 50 504 573 2000 85.8% chr11 + 85878499 85878568 70 browser details YourSeq 49 456 572 2000 86.6% chr17 - 24670853 24671089 237 browser details YourSeq 49 464 575 2000 85.6% chr14 + 50769789 50770293 505 browser details YourSeq 45 530 583 2000 92.6% chr7 + 81486919 81486973 55 browser details YourSeq 45 514 579 2000 84.9% chr10 + 91055586 91055652 67 browser details YourSeq 43 514 566 2000 90.6% chr14 - 50848628 50848680 53 browser details YourSeq 43 513 569 2000 87.8% chr11 - 73259085 73259141 57 browser details YourSeq 43 499 573 2000 78.7% chr1 + 153433682 153433756 75 browser details YourSeq 42 524 575 2000 90.4% chr11 - 46989015 46989066 52 browser details YourSeq 42 514 573 2000 85.0% chr1 - 177290647 177290706 60 browser details YourSeq 42 520 576 2000 87.8% chr18 + 65513974 65514031 58 browser details YourSeq 42 502 575 2000 81.7% chr12 + 3394856 3394925 70 Note: The 2000 bp section upstream of Exon 4 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% chr7 - 133731968 133733967 2000 browser details YourSeq 226 318 702 2000 87.5% chr9 + 40727686 40932550 204865 browser details YourSeq 226 316 705 2000 84.3% chr5 + 149093910 149094299 390 browser details YourSeq 212 335 715 2000 85.7% chr1 + 36752936 36753297 362 browser details YourSeq 205 331 947 2000 82.4% chr8 + 11306186 11306690 505 browser details YourSeq 203 322 705 2000 86.7% chr17 - 68007997 68008382 386 browser details YourSeq 202 324 994 2000 82.4% chr2 + 33675148 33675656 509 browser details YourSeq 201 316 705 2000 87.9% chr11 + 50838727 50839123 397 browser details YourSeq 200 332 711 2000 86.1% chr12 - 17560316 17560684 369 browser details YourSeq 194 342 705 2000 79.5% chr3 - 102037720 102038082 363 browser details YourSeq 193 317 704 2000 84.5% chr18 - 56364306 56364683 378 browser details YourSeq 192 318 683 2000 87.1% chr7 + 129637551 129637919 369 browser details YourSeq 192 322 694 2000 86.1% chr15 + 31666758 31667133 376 browser details YourSeq 192 322 705 2000 84.0% chr12 + 75863115 75863499 385 browser details YourSeq 190 331 693 2000 88.8% chr5 + 37182465 37182828 364 browser details YourSeq 190 342 704 2000 89.4% chr10 + 119689996 119815299 125304 browser details YourSeq 188 318 679 2000 87.5% chr18 + 74968906 74969273 368 browser details YourSeq 187 370 687 2000 84.4% chr7 - 37520957 37521273 317 browser details YourSeq 186 219 697 2000 80.2% chr18 - 66173893 66174593 701 browser details YourSeq 184 404 704 2000 80.4% chr10 - 116624598 116624894 297 Note: The 2000 bp section downstream of Exon 7 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Dhx32 DEAH (Asp-Glu-Ala-His) box polypeptide 32 [ Mus musculus (house mouse) ] Gene ID: 101437, updated on 10-Oct-2019 Gene summary Official Symbol Dhx32 provided by MGI Official Full Name DEAH (Asp-Glu-Ala-His) box polypeptide 32 provided by MGI Primary source MGI:MGI:2141813 See related Ensembl:ENSMUSG00000030986 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 Ddx32; muDDX32; AA408140; 3110079L04Rik; 4732469F02Rik Expression Ubiquitous expression in subcutaneous fat pad adult (RPKM 23.6), placenta adult (RPKM 18.1) and 28 other tissues See Orthologs more human all Genomic context Location: 7; 7 F3 See Dhx32 in Genome Data Viewer Exon count: 14 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 7 NC_000073.6 (133720935..133782785, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 7 NC_000073.5 (140912618..140968486, complement) Chromosome 7 - NC_000073.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 10 transcripts Gene: Dhx32 ENSMUSG00000030986 Description DEAH (Asp-Glu-Ala-His) box polypeptide 32 [Source:MGI Symbol;Acc:MGI:2141813] Gene Synonyms Ddx32 Location Chromosome 7: 133,720,942-133,782,726 reverse strand. GRCm38:CM001000.2 About this gene This gene has 10 transcripts (splice variants), 242 orthologues, 18 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Dhx32-201 ENSMUST00000033290.11 2955 751aa ENSMUSP00000033290.5 Protein coding CCDS21936 Q8BZS9 TSL:1 GENCODE basic APPRIS P1 Dhx32-202 ENSMUST00000063669.7 2644 751aa ENSMUSP00000066067.1 Protein coding CCDS21936 Q8BZS9 TSL:1 GENCODE basic APPRIS P1 Dhx32-203 ENSMUST00000106139.7 2233 611aa ENSMUSP00000101745.1 Protein coding CCDS72060 D3Z4E3 TSL:1 GENCODE basic Dhx32-204 ENSMUST00000130182.1 769 110aa ENSMUSP00000115677.1 Protein coding - D3YZD7 CDS 3' incomplete TSL:3 Dhx32-205 ENSMUST00000135989.1 604 8aa ENSMUSP00000118326.1 Protein coding - A0A1C7ZMZ3 CDS 3' incomplete TSL:3 Dhx32-209 ENSMUST00000149876.1 558 143aa ENSMUSP00000121789.1 Protein coding - D3Z255 CDS 3' incomplete TSL:2 Dhx32-208 ENSMUST00000146211.1 546 No protein - Retained intron - - TSL:1 Dhx32-207 ENSMUST00000140593.8 2998 No protein - lncRNA - - TSL:5 Dhx32-206 ENSMUST00000136301.1 698 No protein - lncRNA - - TSL:2 Dhx32-210 ENSMUST00000211450.1 673 No protein - lncRNA - - TSL:5 Page 7 of 9 https://www.alphaknockout.com 81.78 kb Forward strand Genes Bccip-201 >protein coding Gm45670-201 >lncRNA Fank1-201 >protein coding (Comprehensive set... Bccip-205 >retained intron Fank1-204 >protein coding Bccip-202 >retained intron Fank1-203 >protein coding Bccip-204 >lncRNA Fank1-202 >protein coding Bccip-203 >retained intron Fank1-205 >protein coding Contigs < AC127348.4 Genes (Comprehensive set... < Dhx32-201protein coding < Dhx32-207lncRNA < Dhx32-203protein coding < Dhx32-202protein coding < Dhx32-206lncRNA < Gm15483-201processed pseudogene < Dhx32-205protein coding < Dhx32-210lncRNA < Dhx32-208retained intron < Dhx32-209protein coding < Dhx32-204protein coding Regulatory Build Reverse strand 81.78 kb Regulation Legend CTCF Enhancer Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding RNA gene pseudogene processed transcript Page 8 of 9 https://www.alphaknockout.com Transcript: ENSMUST00000033290 < Dhx32-201protein coding Reverse strand 55.86 kb ENSMUSP00000033... MobiDB lite Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase SMART Helicase-associated domain Pfam Helicase-associated domain Domain of unknown function DUF1605 PANTHER PTHR18934 PTHR18934:SF88 Gene3D 3.40.50.300 1.20.120.1080 CDD cd17977 cd18791 All sequence SNPs/i..
Recommended publications
  • Genome-Wide DNA Methylation Map of Human Neutrophils Reveals Widespread Inter-Individual Epigenetic Variation
    www.nature.com/scientificreports OPEN Genome-wide DNA methylation map of human neutrophils reveals widespread inter-individual Received: 15 June 2015 Accepted: 29 October 2015 epigenetic variation Published: 27 November 2015 Aniruddha Chatterjee1,2, Peter A. Stockwell3, Euan J. Rodger1, Elizabeth J. Duncan2,4, Matthew F. Parry5, Robert J. Weeks1 & Ian M. Morison1,2 The extent of variation in DNA methylation patterns in healthy individuals is not yet well documented. Identification of inter-individual epigenetic variation is important for understanding phenotypic variation and disease susceptibility. Using neutrophils from a cohort of healthy individuals, we generated base-resolution DNA methylation maps to document inter-individual epigenetic variation. We identified 12851 autosomal inter-individual variably methylated fragments (iVMFs). Gene promoters were the least variable, whereas gene body and upstream regions showed higher variation in DNA methylation. The iVMFs were relatively enriched in repetitive elements compared to non-iVMFs, and were associated with genome regulation and chromatin function elements. Further, variably methylated genes were disproportionately associated with regulation of transcription, responsive function and signal transduction pathways. Transcriptome analysis indicates that iVMF methylation at differentially expressed exons has a positive correlation and local effect on the inclusion of that exon in the mRNA transcript. Methylation of DNA is a mechanism for regulating gene function in all vertebrates. It has a role in gene silencing, tissue differentiation, genomic imprinting, chromosome X inactivation, phenotypic plasticity, and disease susceptibility1,2. Aberrant DNA methylation has been implicated in the pathogenesis of sev- eral human diseases, especially cancer3–5. Variation in DNA methylation patterns in healthy individuals has been hypothesised to alter human phenotypes including susceptibility to common diseases6 and response to drug treatments7.
    [Show full text]
  • Identification of Inherited Retinal Disease-Associated Genetic Variants in 11 Candidate Genes
    G C A T T A C G G C A T genes Article Identification of Inherited Retinal Disease-Associated Genetic Variants in 11 Candidate Genes Galuh D. N. Astuti 1,2, L. Ingeborgh van den Born 3, M. Imran Khan 1,4, Christian P. Hamel 5,6,7,†, Béatrice Bocquet 5,6,7, Gaël Manes 5,6, Mathieu Quinodoz 8, Manir Ali 9 ID , Carmel Toomes 9, Martin McKibbin 10 ID , Mohammed E. El-Asrag 9,11, Lonneke Haer-Wigman 1, Chris F. Inglehearn 9 ID , Graeme C. M. Black 12, Carel B. Hoyng 13, Frans P. M. Cremers 1,4 and Susanne Roosing 1,4,* ID 1 Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands; [email protected] (G.D.N.A.); [email protected] (M.I.K.); [email protected] (L.H.-W.); [email protected] (F.P.M.C.) 2 Radboud Institute for Molecular Life Sciences, Radboud University, 6525 GA Nijmegen, The Netherlands 3 The Rotterdam Eye Hospital, 3011 BH Rotterdam, The Netherlands; [email protected] 4 Donders Institute for Brain, Cognition and Behaviour, Radboud University Nijmegen, 6525 EN Nijmegen, The Netherlands 5 Institut National de la Santé et de la Recherche Médicale, Institute for Neurosciences of Montpellier, 34080 Montpellier, France; [email protected] (B.B.); [email protected] (G.M.) 6 University of Montpellier, 34090 Montpellier, France 7 CHRU, Genetics of Sensory Diseases, 34295 Montpellier, France 8 Department of Computational Biology, Unit of Medical Genetics, University of Lausanne, 1015 Lausanne, Switzerland; [email protected] 9 Section of Ophthalmology & Neuroscience, Leeds Institute of Biomedical & Clinical Sciences, University of Leeds, St.
    [Show full text]
  • Genome-Wide Linkage and Association Study Implicates the 10Q26 Region As a Major Genetic Contributor to Primary Nonsyndromic
    www.nature.com/scientificreports OPEN Genome-wide linkage and association study implicates the 10q26 region as a major Received: 6 July 2017 Accepted: 6 October 2017 genetic contributor to primary Published: xx xx xxxx nonsyndromic vesicoureteric refux John M. Darlow1,2, Rebecca Darlay3, Mark G. Dobson1,2, Aisling Stewart3, Pimphen Charoen4,5, Jennifer Southgate 6, Simon C. Baker 6, Yaobo Xu3, Manuela Hunziker2,7, Heather J. Lambert8, Andrew J. Green1,9, Mauro Santibanez-Koref3, John A. Sayer 3, Timothy H. J. Goodship3, Prem Puri2,10, Adrian S. Woolf 11,12, Rajko B. Kenda13, David E. Barton1,9 & Heather J. Cordell3 Vesicoureteric refux (VUR) is the commonest urological anomaly in children. Despite treatment improvements, associated renal lesions – congenital dysplasia, acquired scarring or both – are a common cause of childhood hypertension and renal failure. Primary VUR is familial, with transmission rate and sibling risk both approaching 50%, and appears highly genetically heterogeneous. It is often associated with other developmental anomalies of the urinary tract, emphasising its etiology as a disorder of urogenital tract development. We conducted a genome-wide linkage and association study in three European populations to search for loci predisposing to VUR. Family-based association analysis of 1098 parent-afected-child trios and case/control association analysis of 1147 cases and 3789 controls did not reveal any compelling associations, but parametric linkage analysis of 460 families (1062 afected individuals) under a dominant model identifed a single region, on 10q26, that showed strong linkage (HLOD = 4.90; ZLRLOD = 4.39) to VUR. The ~9Mb region contains 69 genes, including some good biological candidates.
    [Show full text]
  • DEAH-Box Polypeptide 32 Promotes Hepatocellular Carcinoma Progression Via Activating the Β-Catenin Pathway
    DEAH-box polypeptide 32 promotes hepatocellular carcinoma progression via activating the β-catenin pathway Xiaoyun Hu Southern Medical University Nanfang Hospital Guosheng Yuan Southern Medical University Nanfang Hospital Qi Li Southern Medical University Nanfang Hospital Jing Huang Southern Medical University Nanfang Hospital Xiao Cheng Southern Medical University Nanfang Hospital Jinzhang Chen ( [email protected] ) Southern Medical University Nanfang Hospital https://orcid.org/0000-0003-4881-4663 Primary research Keywords: hepatocellular carcinoma, DHX32, EMT, proliferation, β-catenin Posted Date: May 15th, 2020 DOI: https://doi.org/10.21203/rs.3.rs-27420/v1 License: This work is licensed under a Creative Commons Attribution 4.0 International License. Read Full License Page 1/18 Abstract Background Hepatocellular carcinoma (HCC) is a refractory cancer with high morbidity and high mortality. It has been reported that DEAH-box polypeptide 32 (DHX32) was upregulated in several types of malignancies and predicted poor prognosis, which was associated with tumor growth and metastasis. However, the expression of DHX32 in HCC and its role in HCC progression remain largely unknown. Methods Western blot and RT-PCR assays were used to detect the expression of DHX32 and epithelial mesenchymal transition (EMT)-related genes in HCC cells. Wound-healing and Transwell invasion assays were performed to determine the effect of DHX32 and β-catenin on the migration and invasion of HCC cells. Cell proliferation was examined by EdU cell proliferation assay. Results In our study, we found that high level of DHX32 expression was associated with reduced overall survival in HCC patients. DHX32 expression was upregulated in human HCC cells and ectopic expression of DHX32 induced EMT, promoted the migration, invasion, and proliferation of HCC cells, and enhanced tumor growth.
    [Show full text]
  • DHX32 Expression Suggests a Role in Lymphocyte Differentiation
    ANTICANCER RESEARCH 25: 2645-2648 (2005) DHX32 Expression Suggests a Role in Lymphocyte Differentiation MOHAMED ABDELHALEEM, TIE-HUA SUN and MICHAEL HO Department of Paediatric Laboratory Medicine, Hospital for Sick Children, University of Toronto, Canada Abstract. RNA helicases constitute a large group of enzymes gene family, whose members have an RNA helicase-like involved in all aspects of RNA metabolism. Several RNA motif, have been shown to be involved in lymphocyte helicases are dysregulated in cancer, whereas several others are development and activation (9). These findings raise the involved in differentiation. DHX32 has previously been possibility that other RNA helicases might be involved in identified as a novel RNA helicase with a unique structure and hematopoiesis. expression pattern. DHX32 message was down-regulated in DHX32 has been have identified as a novel RNA helicase acute lymphoblastic leukemia cell lines and patient samples. gene which is down regulated in Acute Lymphoblastic In this report, anti-DHX32 was used to study its expression in Leukemia (10). The gene designation was changed to thymus. Immunohistochemistry and flow cytometry showed DHX32, according to the new nomenclature of RNA positive correlation of DHX32 expression with thymocyte helicases (3), to reflect its homology to the DHX family of maturation. These results suggest that DHX32 might play a helicases. Since blasts in precursor acute lymphoblastic role in normal lymphocyte differentiation. leukemia can represent early stages of lymphocyte differentiation, the possibility that DHX32 expression is RNA helicases constitute a large group of conserved correlated with normal lymphocyte differentiation was enzymes characterized by the presence of a centrally located investigated.
    [Show full text]
  • DEAH)/RNA Helicase a Helicases Sense Microbial DNA in Human Plasmacytoid Dendritic Cells
    Aspartate-glutamate-alanine-histidine box motif (DEAH)/RNA helicase A helicases sense microbial DNA in human plasmacytoid dendritic cells Taeil Kima, Shwetha Pazhoora, Musheng Baoa, Zhiqiang Zhanga, Shino Hanabuchia, Valeria Facchinettia, Laura Bovera, Joel Plumasb, Laurence Chaperotb, Jun Qinc, and Yong-Jun Liua,1 aDepartment of Immunology, Center for Cancer Immunology Research, University of Texas M. D. Anderson Cancer Center, Houston, TX 77030; bDepartment of Research and Development, Etablissement Français du Sang Rhône-Alpes Grenoble, 38701 La Tronche, France; and cDepartment of Biochemistry, Baylor College of Medicine, Houston, TX 77030 Edited by Ralph M. Steinman, The Rockefeller University, New York, NY, and approved July 14, 2010 (received for review May 10, 2010) Toll-like receptor 9 (TLR9) senses microbial DNA and triggers type I Microbial nucleic acids, including their genomic DNA/RNA IFN responses in plasmacytoid dendritic cells (pDCs). Previous and replicating intermediates, work as strong PAMPs (13), so studies suggest the presence of myeloid differentiation primary finding PRR-sensing pathogenic nucleic acids and investigating response gene 88 (MyD88)-dependent DNA sensors other than their signaling pathway is of general interest. Cytosolic RNA is TLR9 in pDCs. Using MS, we investigated C-phosphate-G (CpG)- recognized by RLRs, including RIG-I, melanoma differentiation- binding proteins from human pDCs, pDC-cell lines, and interferon associated gene 5 (MDA5), and laboratory of genetics and physi- regulatory factor 7 (IRF7)-expressing B-cell lines. CpG-A selectively ology 2 (LGP2). RIG-I senses 5′-triphosphate dsRNA and ssRNA bound the aspartate-glutamate-any amino acid-aspartate/histi- or short dsRNA with blunt ends.
    [Show full text]
  • Genome Provides Insights Into Vertebrate Evolution
    ARTICLES OPEN Sequencing of the sea lamprey (Petromyzon marinus) genome provides insights into vertebrate evolution Jeramiah J Smith1,2, Shigehiro Kuraku3,4, Carson Holt5,37, Tatjana Sauka-Spengler6,37, Ning Jiang7, Michael S Campbell5, Mark D Yandell5, Tereza Manousaki4, Axel Meyer4, Ona E Bloom8,9, Jennifer R Morgan10, Joseph D Buxbaum11–14, Ravi Sachidanandam11, Carrie Sims15, Alexander S Garruss15, Malcolm Cook15, Robb Krumlauf15,16, Leanne M Wiedemann15,17, Stacia A Sower18, Wayne A Decatur18, Jeffrey A Hall18, Chris T Amemiya2,19, Nil R Saha2, Katherine M Buckley20,21, Jonathan P Rast20,21, Sabyasachi Das22,23, Masayuki Hirano22,23, Nathanael McCurley22,23, Peng Guo22,23, Nicolas Rohner24, Clifford J Tabin24, Paul Piccinelli25, Greg Elgar25, Magali Ruffier26, Bronwen L Aken26, Stephen M J Searle26, Matthieu Muffato27, Miguel Pignatelli27, Javier Herrero27, Matthew Jones6, C Titus Brown28,29, Yu-Wen Chung-Davidson30, Kaben G Nanlohy30, Scot V Libants30, Chu-Yin Yeh30, David W McCauley31, James A Langeland32, Zeev Pancer33, Bernd Fritzsch34, Pieter J de Jong35, Baoli Zhu35,37, Lucinda L Fulton36, Brenda Theising36, Paul Flicek27, Marianne E Bronner6, All rights reserved. Wesley C Warren36, Sandra W Clifton36,37, Richard K Wilson36 & Weiming Li30 Lampreys are representatives of an ancient vertebrate lineage that diverged from our own ~500 million years ago. By virtue of this deeply shared ancestry, the sea lamprey (P. marinus) genome is uniquely poised to provide insight into the ancestry of vertebrate genomes and the underlying principles of vertebrate biology. Here, we present the first lamprey whole-genome sequence and America, Inc. assembly. We note challenges faced owing to its high content of repetitive elements and GC bases, as well as the absence of broad-scale sequence information from closely related species.
    [Show full text]
  • The Pdx1 Bound Swi/Snf Chromatin Remodeling Complex Regulates Pancreatic Progenitor Cell Proliferation and Mature Islet Β Cell
    Page 1 of 125 Diabetes The Pdx1 bound Swi/Snf chromatin remodeling complex regulates pancreatic progenitor cell proliferation and mature islet β cell function Jason M. Spaeth1,2, Jin-Hua Liu1, Daniel Peters3, Min Guo1, Anna B. Osipovich1, Fardin Mohammadi3, Nilotpal Roy4, Anil Bhushan4, Mark A. Magnuson1, Matthias Hebrok4, Christopher V. E. Wright3, Roland Stein1,5 1 Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, TN 2 Present address: Department of Pediatrics, Indiana University School of Medicine, Indianapolis, IN 3 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 4 Diabetes Center, Department of Medicine, UCSF, San Francisco, California 5 Corresponding author: [email protected]; (615)322-7026 1 Diabetes Publish Ahead of Print, published online June 14, 2019 Diabetes Page 2 of 125 Abstract Transcription factors positively and/or negatively impact gene expression by recruiting coregulatory factors, which interact through protein-protein binding. Here we demonstrate that mouse pancreas size and islet β cell function are controlled by the ATP-dependent Swi/Snf chromatin remodeling coregulatory complex that physically associates with Pdx1, a diabetes- linked transcription factor essential to pancreatic morphogenesis and adult islet-cell function and maintenance. Early embryonic deletion of just the Swi/Snf Brg1 ATPase subunit reduced multipotent pancreatic progenitor cell proliferation and resulted in pancreas hypoplasia. In contrast, removal of both Swi/Snf ATPase subunits, Brg1 and Brm, was necessary to compromise adult islet β cell activity, which included whole animal glucose intolerance, hyperglycemia and impaired insulin secretion. Notably, lineage-tracing analysis revealed Swi/Snf-deficient β cells lost the ability to produce the mRNAs for insulin and other key metabolic genes without effecting the expression of many essential islet-enriched transcription factors.
    [Show full text]
  • Cell Type Manuscript Rev 180328 Justmaintext
    Running Head: PREDICTING CELL TYPE BALANCE 1012 7. Supporting Information 1013 1014 1015 Supplementary Material for: 1016 1017 INFERENCE OF CELL TYPE COMPOSITION FROM HUMAN BRAIN TRANSCRIPTOMIC 1018 DATASETS ILLUMINATES THE EFFECTS OF AGE, MANNER OF DEATH, DISSECTION, 1019 AND PSYCHIATRIC DIAGNOSIS 1020 *Megan Hastings Hagenauer, Ph.D.1, Anton Schulmann, M.D.2, Jun Z. Li, Ph.D.3, Marquis P. Vawter, 1021 Ph.D.4, David M. Walsh, Psy.D.4, Robert C. Thompson, Ph.D.1, Cortney A. Turner, Ph.D.1, William E. 1022 Bunney, M.D.4, Richard M. Myers, Ph.D.5, Jack D. Barchas, M.D.6, Alan F. Schatzberg, M.D.7, Stanley J. 1023 Watson, M.D., Ph.D.1, Huda Akil, Ph.D.1 1024 42 Running Head: PREDICTING CELL TYPE BALANCE 1025 7.1 Additional Methods: Detailed Preprocessing Methods for the Transcriptomic Datasets 1026 1027 7.1.1 RNA-Seq data from Purified Cell Types (GSE52564 and GSE67835) 1028 As initial validation, we used our method to predict sample cell type identity in two RNA-Seq 1029 datasets derived from purified cell types: one derived from from purified cortical cell types in mice 1030 (n=17: two samples per cell type and 3 whole brain samples: GSE52564) (18), and one derived from 466 1031 single-cells dissociated from freshly-resected human cortex (GSE67835) (2). The RNA-Seq data that we 1032 downloaded from GEO was already in the format of FPKM values (Fragments Per Kilobase of exon 1033 model per million mapped fragments) (18) or counts per gene (2).
    [Show full text]
  • Overexpression of DHX32 Contributes to the Growth and Metastasis of Colorectal Cancer
    OPEN Overexpression of DHX32 contributes to SUBJECT AREAS: the growth and metastasis of colorectal ONCOGENES CELL SIGNALLING cancer Huayue Lin1*, Wenjuan Liu1*, Zanxi Fang1, Xianming Liang1, Juan Li1, Yongying Bai1, Lingqing Lin1, Received Hanyu You1, Yihua Pei3, Fen Wang4 & Zhong-Ying Zhang1,2 18 September 2014 Accepted 1Center for Clinical Laboratory, Xiamen University Affiliated Zhongshan Hospital, Xiamen, China, 2State Key Laboratory of 25 February 2015 Molecular Vaccinology and Molecular Diagnostics, School of Public Health, Xiamen University, Xiamen, China, 3Central Laboratory, Xiamen University Affiliated Zhongshan Hospital, Xiamen, China, 4Center for Cancer and Stem Cell Biology, Institute of Published Biosciences and Technology, Texas A&M Health Science Center, Houston, TX. 18 March 2015 Our previous work demonstrates that DHX32 is upregulated in colorectal cancer (CRC) compared to its Correspondence and adjacent normal tissues. However, how overexpressed DHX32 contributes to CRC remains largely unknown. In this study, we reported that DHX32 was overexpressed in human colon cancer cells. requests for materials Overexpressed DHX32 promoted SW480 cancer cells proliferation, migration, and invasion, as well as should be addressed to decreased the susceptibility to chemotherapy agent 5-Fluorouracil. Furthermore, PCR array analyses F.W. ([email protected]. revealed that depleting DHX32 in SW480 colon cancer cells suppressed expression of WISP1, MMP7 and edu) or Z.-Y.Z. VEGFA in the Wnt pathway, and anti-apoptotic gene BCL2 and CA9, however, elevated expression of (zzy11603@163. pro-apoptotic gene ACSL5. The findings suggested that overexpressed DHX32 played an important role in com) CRC progression and metastasis and that DHX32 has the potential to serve as a biomarker and a novel therapeutic target for CRC.
    [Show full text]
  • CRISPR-Cas9 Genome Editing Induces Megabase-Scale Chromosomal Truncations
    ARTICLE https://doi.org/10.1038/s41467-019-09006-2 OPEN CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations Grégoire Cullot1,2, Julian Boutin1,2,3, Jérôme Toutain4, Florence Prat1,2, Perrine Pennamen4, Caroline Rooryck4, Martin Teichmann1,5, Emilie Rousseau1,5, Isabelle Lamrissi-Garcia1,2, Véronique Guyonnet-Duperat2,6, Alice Bibeyran2,6, Magalie Lalanne1,2, Valérie Prouzet-Mauléon1,7, Béatrice Turcq1,7, Cécile Ged1,2,3,8, Jean-Marc Blouin1,2,3,8, Emmanuel Richard1,2,3,8, Sandrine Dabernat1,2,3, François Moreau-Gaudry1,2,3,6,8 & Aurélie Bedel1,2,3,8 1234567890():,; CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease- induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal trun- cations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies. 1 Univ. Bordeaux, 33000 Bordeaux, France. 2 INSERM U1035, Biotherapy of genetic diseases, inflammatory disorders and cancers, 33000 Bordeaux, France.
    [Show full text]
  • Inference of Cell Type Composition from Human Brain Transcriptomic
    bioRxiv preprint doi: https://doi.org/10.1101/089391; this version posted December 20, 2017. 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. Running Head: PREDICTING CELL TYPE BALANCE INFERENCE OF CELL TYPE COMPOSITION FROM HUMAN BRAIN TRANSCRIPTOMIC DATASETS ILLUMINATES THE EFFECTS OF AGE, MANNER OF DEATH, DISSECTION, AND PSYCHIATRIC DIAGNOSIS *Megan Hastings Hagenauer, Ph.D.1, Anton Schulmann, M.D.2, Jun Z. Li, Ph.D.3, Marquis P. Vawter, Ph.D.4, David M. Walsh, Psy.D.4, Robert C. Thompson, Ph.D.1, Cortney A. Turner, Ph.D.1, William E. Bunney, M.D.4, Richard M. Myers, Ph.D.5, Jack D. Barchas, M.D.6, Alan F. Schatzberg, M.D.7, Stanley J. Watson, M.D., Ph.D.1, Huda Akil, Ph.D.1 1Mol. Behavioral Neurosci. Inst., Univ. of Michigan, Ann Arbor, MI, USA; 2 Janelia Research Campus, Howard Hughes Medical Institute, Ashburn, VA, USA, 3Genet., Univ. of Michigan, Ann Arbor, MI, USA; 4Univ. of California, Irvine, CA; 5HudsonAlpha Inst. for Biotech., Huntsville, AL, USA; 6Stanford, Palo Alto, CA, 7Cornell, New York, NY, USA *Corresponding Author: Megan Hastings Hagenauer, Ph.D. e-mail: [email protected] Molecular Behavioral Neuroscience Institute (MBNI) 205 Zina Pitcher Pl. Ann Arbor, MI 48109 1 bioRxiv preprint doi: https://doi.org/10.1101/089391; this version posted December 20, 2017. 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.
    [Show full text]