DOCSLIB.ORG

Mouse Naa15 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Naa15 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Naa15 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Naa15 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Naa15 gene (NCBI Reference Sequence: NM_053089 ; Ensembl: ENSMUSG00000063273 ) is located on Mouse chromosome 3. 20 exons are identified, with the ATG start codon in exon 1 and the TGA stop codon in exon 20 (Transcript: ENSMUST00000029303). Exon 4~5 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Naa15 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-407I18 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 4 starts from about 9.44% of the coding region. The knockout of Exon 4~5 will result in frameshift of the gene. The size of intron 3 for 5'-loxP site insertion: 3462 bp, and the size of intron 5 for 3'-loxP site insertion: 927 bp. The size of effective cKO region: ~1465 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 5' gRNA region gRNA region 3' 1 4 5 6 20 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Naa15 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. Tandem repeats are found in the dot plot matrix. It may be difficult to construct this targeting vector. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(7965bp) | A(28.64% 2281) | C(16.28% 1297) | T(34.6% 2756) | G(20.48% 1631) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 8 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% chr3 + 51438657 51441656 3000 browser details YourSeq 280 2129 2914 3000 85.4% chr13 + 59698239 59698803 565 browser details YourSeq 265 2128 2921 3000 86.3% chr11 - 106447215 106447964 750 browser details YourSeq 249 2625 2918 3000 92.9% chr11 - 19857153 19886150 28998 browser details YourSeq 246 2179 2918 3000 85.2% chr18 - 42238275 42238794 520 browser details YourSeq 239 2623 2918 3000 92.9% chr1 - 86513304 86513600 297 browser details YourSeq 236 2625 2918 3000 91.3% chr18 - 62612540 62612836 297 browser details YourSeq 236 2624 2917 3000 92.7% chr13 - 114065687 114065978 292 browser details YourSeq 235 2626 2918 3000 91.4% chr4 - 58921792 58922082 291 browser details YourSeq 235 2625 2926 3000 90.5% chr2 + 142896398 142896703 306 browser details YourSeq 234 2624 2918 3000 92.2% chr8 - 42834484 42834791 308 browser details YourSeq 233 2624 2918 3000 90.9% chr15 + 99679926 99680220 295 browser details YourSeq 232 2624 2918 3000 91.1% chr8 + 11358744 11359037 294 browser details YourSeq 230 2625 2923 3000 90.3% chr12 + 100158117 100158413 297 browser details YourSeq 229 2624 2918 3000 91.3% chr5 - 44026949 44027242 294 browser details YourSeq 229 2624 2918 3000 92.7% chr15 - 11173372 11173677 306 browser details YourSeq 229 2625 2918 3000 90.6% chr3 + 32055111 32055402 292 browser details YourSeq 229 2624 2918 3000 88.9% chr11 + 104276922 104277210 289 browser details YourSeq 229 2628 2918 3000 91.4% chr11 + 53212058 53212344 287 browser details YourSeq 228 2625 2919 3000 90.9% chr19 + 58384319 58384611 293 Note: The 3000 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 3000 1 3000 3000 100.0% chr3 + 51443122 51446121 3000 browser details YourSeq 60 2228 2310 3000 92.9% chr15 - 81818746 81818834 89 browser details YourSeq 59 2228 2310 3000 95.4% chr9 - 16929446 16929534 89 browser details YourSeq 59 2191 2312 3000 90.3% chr12 - 110592906 110593029 124 browser details YourSeq 58 2216 2310 3000 88.2% chr9 - 38791676 38791776 101 browser details YourSeq 58 2182 2300 3000 92.7% chr17 + 82887271 82887391 121 browser details YourSeq 57 2228 2310 3000 93.9% chr7 - 90613756 90613844 89 browser details YourSeq 57 2228 2299 3000 92.6% chr15 - 98957057 98957136 80 browser details YourSeq 56 2237 2313 3000 93.8% chr13 - 48623940 48624022 83 browser details YourSeq 55 2228 2313 3000 92.4% chr7 + 138888516 138888607 92 browser details YourSeq 55 2237 2310 3000 95.1% chr14 + 58568157 58568236 80 browser details YourSeq 55 2237 2310 3000 93.7% chr11 + 96136871 96136954 84 browser details YourSeq 53 2238 2304 3000 83.9% chr11 + 75935317 75935378 62 browser details YourSeq 52 2237 2312 3000 90.7% chr13 - 103477126 103477207 82 browser details YourSeq 52 2228 2299 3000 93.4% chr5 + 108633306 108633383 78 browser details YourSeq 52 2228 2299 3000 93.4% chr13 + 20004966 20005043 78 browser details YourSeq 50 2228 2300 3000 94.7% chr1 - 123121413 123121491 79 browser details YourSeq 49 2228 2300 3000 94.6% chr17 - 93867511 93867589 79 browser details YourSeq 48 2240 2300 3000 92.9% chr17 + 69544367 69544431 65 browser details YourSeq 48 2268 2322 3000 94.6% chr13 + 69350000 69350055 56 Note: The 3000 bp section downstream of Exon 5 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 https://www.alphaknockout.com Gene and protein information: Naa15 N(alpha)-acetyltransferase 15, NatA auxiliary subunit [ Mus musculus (house mouse) ] Gene ID: 74838, updated on 12-Aug-2019 Gene summary Official Symbol Naa15 provided by MGI Official Full Name N(alpha)-acetyltransferase 15, NatA auxiliary subunit provided by MGI Primary source MGI:MGI:1922088 See related Ensembl:ENSMUSG00000063273 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 Narg1; mNAT1; ASTBDN; Tbdn-1; 6330400I15; 5730450D16Rik Expression Broad expression in CNS E11.5 (RPKM 15.4), placenta adult (RPKM 10.3) and 22 other tissues See more Orthologs human all Genomic context Location: 3; 3 C See Naa15 in Genome Data Viewer Exon count: 20 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 3 NC_000069.6 (51416016..51475985) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 3 NC_000069.5 (51219938..51279907) Chromosome 3 - NC_000069.6 Page 5 of 8 https://www.alphaknockout.com Transcript information: This gene has 9 transcripts Gene: Naa15 ENSMUSG00000063273 Description N(alpha)-acetyltransferase 15, NatA auxiliary subunit [Source:MGI Symbol;Acc:MGI:1922088] Gene Synonyms 5730450D16Rik, ASTBDN, Narg1, Tbdn-1, mNAT1, tubedown Location Chromosome 3: 51,415,148-51,476,507 forward strand. GRCm38:CM000996.2 About this gene This gene has 9 transcripts (splice variants), 258 orthologues, 2 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Naa15- ENSMUST00000029303.12 6090 865aa ENSMUSP00000029303.7 Protein coding CCDS17340 G3X8Y3 TSL:1 201 GENCODE basic APPRIS P1 Naa15- ENSMUST00000193266.5 7480 815aa ENSMUSP00000141433.1 Protein coding - A0A0A6YW80 TSL:1 205 GENCODE basic Naa15- ENSMUST00000192197.1 712 237aa ENSMUSP00000141886.1 Protein coding - A0A0A6YX86 CDS 5' and 3' 202 incomplete TSL:3 Naa15- ENSMUST00000192419.5 637 126aa ENSMUSP00000141965.1 Protein coding - A0A0A6YXF4 CDS 3' incomplete 203 TSL:5 Naa15- ENSMUST00000193267.1 3073 No - Retained - - TSL:NA 206 protein intron Naa15- ENSMUST00000195430.1 2975 No - Retained - - TSL:NA 209 protein intron Naa15- ENSMUST00000194685.1 2319 No - Retained - - TSL:NA 208 protein intron Naa15- ENSMUST00000193694.1 667 No - Retained - - TSL:3 207 protein intron Naa15- ENSMUST00000192523.5 629 No - lncRNA - - TSL:3 204 protein Page 6 of 8 https://www.alphaknockout.com 81.36 kb Forward strand Genes (Comprehensive set... Naa15-205 >protein coding Rab33b-203 >lncRNA Naa15-201 >protein coding Naa15-204 >lncRNA Rab33b-202 >protein coding Naa15-203 >protein coding Naa15-207 >retained intron Rab33b-201 >protein coding Gm37646-201 >TEC Naa15-209 >retained intron Naa15-208 >retained intron Naa15-202 >protein coding Naa15-206 >retained intron Contigs AC105966.11 > AC102860.13 > Genes < Ndufc1-201protein coding < Gm38160-201TEC (Comprehensive set... < Ndufc1-203retained intron < Ndufc1-202lncRNA < Ndufc1-204protein coding Regulatory Build Reverse strand 81.36 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 processed transcript RNA gene Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000029303
Load more

Recommended publications
  • Supp Material.Pdf
    Simon et al. Supplementary information: Table of contents p.1 Supplementary material and methods p.2-4 • PoIy(I)-poly(C) Treatment • Flow Cytometry and Immunohistochemistry • Western Blotting • Quantitative RT-PCR • Fluorescence In Situ Hybridization • RNA-Seq • Exome capture • Sequencing Supplementary Figures and Tables Suppl. items Description pages Figure 1 Inactivation of Ezh2 affects normal thymocyte development 5 Figure 2 Ezh2 mouse leukemias express cell surface T cell receptor 6 Figure 3 Expression of EZH2 and Hox genes in T-ALL 7 Figure 4 Additional mutation et deletion of chromatin modifiers in T-ALL 8 Figure 5 PRC2 expression and activity in human lymphoproliferative disease 9 Figure 6 PRC2 regulatory network (String analysis) 10 Table 1 Primers and probes for detection of PRC2 genes 11 Table 2 Patient and T-ALL characteristics 12 Table 3 Statistics of RNA and DNA sequencing 13 Table 4 Mutations found in human T-ALLs (see Fig. 3D and Suppl. Fig. 4) 14 Table 5 SNP populations in analyzed human T-ALL samples 15 Table 6 List of altered genes in T-ALL for DAVID analysis 20 Table 7 List of David functional clusters 31 Table 8 List of acquired SNP tested in normal non leukemic DNA 32 1 Simon et al. Supplementary Material and Methods PoIy(I)-poly(C) Treatment. pIpC (GE Healthcare Lifesciences) was dissolved in endotoxin-free D-PBS (Gibco) at a concentration of 2 mg/ml. Mice received four consecutive injections of 150 μg pIpC every other day. The day of the last pIpC injection was designated as day 0 of experiment.
    [Show full text]
  • Supplementary Table S4. FGA Co-Expressed Gene List in LUAD
    Supplementary Table S4. FGA co-expressed gene list in LUAD tumors Symbol R Locus Description FGG 0.919 4q28 fibrinogen gamma chain FGL1 0.635 8p22 fibrinogen-like 1 SLC7A2 0.536 8p22 solute carrier family 7 (cationic amino acid transporter, y+ system), member 2 DUSP4 0.521 8p12-p11 dual specificity phosphatase 4 HAL 0.51 12q22-q24.1histidine ammonia-lyase PDE4D 0.499 5q12 phosphodiesterase 4D, cAMP-specific FURIN 0.497 15q26.1 furin (paired basic amino acid cleaving enzyme) CPS1 0.49 2q35 carbamoyl-phosphate synthase 1, mitochondrial TESC 0.478 12q24.22 tescalcin INHA 0.465 2q35 inhibin, alpha S100P 0.461 4p16 S100 calcium binding protein P VPS37A 0.447 8p22 vacuolar protein sorting 37 homolog A (S. cerevisiae) SLC16A14 0.447 2q36.3 solute carrier family 16, member 14 PPARGC1A 0.443 4p15.1 peroxisome proliferator-activated receptor gamma, coactivator 1 alpha SIK1 0.435 21q22.3 salt-inducible kinase 1 IRS2 0.434 13q34 insulin receptor substrate 2 RND1 0.433 12q12 Rho family GTPase 1 HGD 0.433 3q13.33 homogentisate 1,2-dioxygenase PTP4A1 0.432 6q12 protein tyrosine phosphatase type IVA, member 1 C8orf4 0.428 8p11.2 chromosome 8 open reading frame 4 DDC 0.427 7p12.2 dopa decarboxylase (aromatic L-amino acid decarboxylase) TACC2 0.427 10q26 transforming, acidic coiled-coil containing protein 2 MUC13 0.422 3q21.2 mucin 13, cell surface associated C5 0.412 9q33-q34 complement component 5 NR4A2 0.412 2q22-q23 nuclear receptor subfamily 4, group A, member 2 EYS 0.411 6q12 eyes shut homolog (Drosophila) GPX2 0.406 14q24.1 glutathione peroxidase
    [Show full text]
  • From Stem Cells to Populations—Using Hipsc, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects
    G C A T T A C G G C A T genes Review From Stem Cells to Populations—Using hiPSC, Next-Generation Sequencing, and GWAS to Explore the Genetic and Molecular Mechanisms of Congenital Heart Defects Martin Broberg 1,2, Johanna Hästbacka 3 and Emmi Helle 1,4,* 1 Stem Cells and Metabolism Research Program, Faculty of Medicine, University of Helsinki, 00290 Helsinki, Finland; martin.broberg@helsinki.fi 2 Institute for Molecular Medicine Finland, HiLIFE, University of Helsinki, 00290 Helsinki, Finland 3 New Children’s Hospital, and Pediatric Research Center, Department of Anesthesia and Intensive Care, Helsinki University Hospital, 00290 Helsinki, Finland; johanna.r.hastbacka@hus.fi 4 New Children’s Hospital, and Pediatric Research Center, Department of Pediatrics, Helsinki University Hospital, 00290 Helsinki, Finland * Correspondence: emmi.helle@helsinki.fi Abstract: Congenital heart defects (CHD) are developmental malformations affecting the heart and the great vessels. Early heart development requires temporally regulated crosstalk between multiple cell types, signaling pathways, and mechanical forces of early blood flow. While both genetic and en- vironmental factors have been recognized to be involved, identifying causal genes in non-syndromic CHD has been difficult. While variants following Mendelian inheritance have been identified by linkage analysis in a few families with multiple affected members, the inheritance pattern in most Citation: Broberg, M.; Hästbacka, J.; familial cases is complex, with reduced penetrance and variable expressivity. Furthermore, most Helle, E. From Stem Cells to non-syndromic CHD are sporadic. Improved sequencing technologies and large biobank collections Populations—Using hiPSC, Next-Generation Sequencing, and have enabled genome-wide association studies (GWAS) in non-syndromic CHD.
    [Show full text]
  • Truncating Variants in NAA15 Are Associated with Variable Levels of Intellectual Disability, Autism Spectrum Disorder, and Congenital Anomalies
    Manuscript Truncating variants in NAA15 are associated with variable levels of intellectual disability, autism spectrum disorder, and congenital anomalies Authors: Hanyin Cheng1*, Avinash V. Dharmadhikari1*, Sylvia Varland2,44, Ning Ma3,4,5, Deepti Domingo6, Robert Kleyner7, Alan F. Rope8, Margaret Yoon7, Asbjørg Stray-Pedersen9,10, Jennifer E. Posey9, Sarah R. Crews11, Mohammad K. Eldomery9, Zeynep Coban Akdemir9, Andrea M. Lewis9,12, Vernon R. Sutton9, Jill A. Rosenfeld9, Erin Conboy13, Katherine Agre13, Fan Xia1,9, Magdalena Walkiewicz1,9,14, Mauro Longoni15,16, Frances A. High15,17,18, Marjon A. van Slegtenhorst19, Grazia M.S. Mancini19, Candice R. Finnila20, Arie van Haeringen21, Nicolette den Hollander21, Claudia Ruivenkamp21, Sakkubai Naidu22, Sonal Mahida22, Elizabeth E. Palmer23,24, Lucinda Murray23, Derek Lim25, Parul Jayakar26, Michael J. Parker27, Stefania Giusto28, Eman- uela Stracuzzi28, Corrado Romano28, Jennifer S. Beighley29, Raphael A. Bernier29, Sébastien Küry30, Mathilde Nizon30, Mark A. Corbett31, Marie Shaw31, Alison Gardner31, Christopher Barnett32, Ruth Armstrong33, Karin S. Kassahn34,35, Anke Van Dijck36, Geert Vandew- eyer36,Tjitske Kleefstra37, Jolanda Schieving37, Marjolijn J. Jongmans37, Bert B.A. de Vries37, Rolph Pfundt37, Bronwyn Kerr38, Samantha K. Rojas39, Kym M. Boycott39, Richard Person40, Re- becca Willaert40, Evan E. Eichler41,42, R. Frank Kooy36, Yaping Yang1,9, Joseph C. Wu3,4,5, James R. Lupski9,43, Thomas Arnesen2,44,45, Gregory M. Cooper20, Wendy K. Chung46, Jozef Gecz6,31,47, Holly A.F. Stessman11,**, Linyan Meng1,9,**, Gholson J. Lyon7,** 1. Baylor Genetics, Houston, TX, 77021, USA; 2. Department of Biomedicine, University of Bergen, N-5020 Bergen, Norway; 3. Stanford Cardiovascular Institute, Stanford University School of Medicine, Stanford, CA 94305, USA; 4.
    [Show full text]
  • Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells
    Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells The MIT Faculty has made this article openly available. Please share how this access benefits you. Your story matters. Citation Gaya, Mauro et al. “Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells.” Cell 172, 3 (January 2018): 517–533 © 2017 The Author(s) As Published http://dx.doi.org/10.1016/j.cell.2017.11.036 Publisher Elsevier Version Final published version Citable link http://hdl.handle.net/1721.1/113555 Terms of Use Creative Commons Attribution 4.0 International License Detailed Terms http://creativecommons.org/licenses/by/4.0/ Article Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells Graphical Abstract Authors Mauro Gaya, Patricia Barral, Marianne Burbage, ..., Andreas Bruckbauer, Jessica Strid, Facundo D. Batista Correspondence [email protected] (M.G.), [email protected] (F.D.B.) In Brief NKT cells are required for the initial formation of germinal centers and production of effective neutralizing antibody responses against viruses. Highlights d NKT cells promote B cell immunity upon viral infection d NKT cells are primed by lymph-node-resident macrophages d NKT cells produce early IL-4 wave at the follicular borders d Early IL-4 wave is required for efficient seeding of germinal centers Gaya et al., 2018, Cell 172, 517–533 January 25, 2018 ª 2017 The Authors. Published by Elsevier Inc. https://doi.org/10.1016/j.cell.2017.11.036 Article Initiation of Antiviral B Cell Immunity Relies on Innate Signals from Spatially Positioned NKT Cells Mauro Gaya,1,2,* Patricia Barral,2,3 Marianne Burbage,2 Shweta Aggarwal,2 Beatriz Montaner,2 Andrew Warren Navia,1,4,5 Malika Aid,6 Carlson Tsui,2 Paula Maldonado,2 Usha Nair,1 Khader Ghneim,7 Padraic G.
    [Show full text]
  • This Item Is the Archived Peer-Reviewed Author-Version Of
    This item is the archived peer-reviewed author-version of: Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases Reference: Stessman Holly A. F., Xiong Bo, Coe Bradley P., Wang Tianyun, Hoekzema Kendra, Fenckova Michaela, Kvarnung Malin, Gerdts Jennifer, Trinh Sandy, Cosemans Nele, ....- Targeted sequencing identifies 91 neurodevelopmental-disorder risk genes with autism and developmental-disability biases Nature genetics - ISSN 1061-4036 - 49:4(2017), p. 515-526 Full text (Publisher's DOI): http://dx.doi.org/doi:10.1038/NG.3792 To cite this reference: http://hdl.handle.net/10067/1424230151162165141 Institutional repository IRUA 1 Targeted sequencing identifies 91 neurodevelopmental disorder risk genes with autism 2 and developmental disability biases 3 4 Holly A. F. Stessman1,Ψ,α, Bo Xiong1,2,Ψ, Bradley P. Coe1,β, Tianyun Wang3,β, Kendra 5 Hoekzema1, Michaela Fenckova4,5, Malin Kvarnung6,7, Jennifer Gerdts8, Sandy Trinh8, Nele 6 Cosemans9, Laura Vives1, Janice Lin1, Tychele N. Turner1, Gijs Santen10, Claudia 7 Ruivenkamp10, Marjolein Kriek10, Arie van Haeringen10, Emmelien Aten10, Kathryn Friend11,12, 8 Jan Liebelt13, Christopher Barnett13, Eric Haan11,13, Marie Shaw11, Jozef Gecz11,14, Britt-Marie 9 Anderlid6,7, Ann Nordgren6,7, Anna Lindstrand6,7, Charles Schwartz15, R. Frank Kooy16, Geert 10 Vandeweyer16, Celine Helsmoortel16, Corrado Romano17, Antonino Alberti17, Mirella Vinci17, 11 Emanuela Avola17, Stefania Giusto18, Eric Courchesne19, Tiziano Pramparo19, Karen Pierce19, 12 Srinivasa Nalabolu19, David Amaral20, Ingrid E. Scheffer21,22,23, Martin B. Delatycki21,24,25, Paul J. 13 Lockhart21,25, Fereydoun Hormozdiari26, Benjamin Harich4,5, Anna Castells-Nobau4,5, Kun Xia3, 14 Hilde Peeters9, Magnus Nordenskjöld6,7, Annette Schenck4,5, Raphael A.
    [Show full text]
  • Understanding the Genetic Basis of Phenotype Variability in Individuals with Neurocognitive Disorders
    Understanding the genetic basis of phenotype variability in individuals with neurocognitive disorders Michael H. Duyzend A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Washington 2016 Reading Committee: Evan E. Eichler, Chair Raphael Bernier Philip Green Program Authorized to Offer Degree: Genome Sciences 1 ©Copyright 2016 Michael H. Duyzend 2 University of Washington Abstract Understanding the genetic basis of phenotype variability in individuals with neurocognitive disorders Michael H. Duyzend Chair of the Supervisory Committee: Professor Evan E. Eichler Department of Genome Sciences Individuals with a diagnosis of a neurocognitive disorder, such as an autism spectrum disorder (ASD), can present with a wide range of phenotypes. Some have severe language and cognitive deficiencies while others are only deficient in social functioning. Sequencing studies have revealed extreme locus heterogeneity underlying the ASDs. Even cases with a known pathogenic variant, such as the 16p11.2 CNV, can be associated with phenotypic heterogeneity. In this thesis, I test the hypothesis that phenotypic heterogeneity observed in populations with a known pathogenic variant, such as the 16p11.2 CNV as well as that associated with the ASDs in general, is due to additional genetic factors. I analyze the phenotypic and genotypic characteristics of over 120 families where at least one individual carries the 16p11.2 CNV, as well as a cohort of over 40 families with high functioning autism and/or intellectual disability. In the 16p11.2 cohort, I assessed variation both internal to and external to the CNV critical region. Among de novo cases, I found a strong maternal bias for the origin of deletions (59/66, 89.4% of cases, p=2.38x10-11), the strongest such effect so far observed for a CNV associated with a microdeletion syndrome, a significant maternal transmission bias for secondary deletions (32 maternal versus 14 paternal, p=1.14x10-2), and nine probands carrying additional CNVs disrupting autism-associated genes.
    [Show full text]
  • Agricultural University of Athens
    ΓΕΩΠΟΝΙΚΟ ΠΑΝΕΠΙΣΤΗΜΙΟ ΑΘΗΝΩΝ ΣΧΟΛΗ ΕΠΙΣΤΗΜΩΝ ΤΩΝ ΖΩΩΝ ΤΜΗΜΑ ΕΠΙΣΤΗΜΗΣ ΖΩΙΚΗΣ ΠΑΡΑΓΩΓΗΣ ΕΡΓΑΣΤΗΡΙΟ ΓΕΝΙΚΗΣ ΚΑΙ ΕΙΔΙΚΗΣ ΖΩΟΤΕΧΝΙΑΣ ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων ΕΙΡΗΝΗ Κ. ΤΑΡΣΑΝΗ ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ: ΑΝΤΩΝΙΟΣ ΚΟΜΙΝΑΚΗΣ ΑΘΗΝΑ 2020 ΔΙΔΑΚΤΟΡΙΚΗ ΔΙΑΤΡΙΒΗ Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων Genome-wide association analysis and gene network analysis for (re)production traits in commercial broilers ΕΙΡΗΝΗ Κ. ΤΑΡΣΑΝΗ ΕΠΙΒΛΕΠΩΝ ΚΑΘΗΓΗΤΗΣ: ΑΝΤΩΝΙΟΣ ΚΟΜΙΝΑΚΗΣ Τριμελής Επιτροπή: Aντώνιος Κομινάκης (Αν. Καθ. ΓΠΑ) Ανδρέας Κράνης (Eρευν. B, Παν. Εδιμβούργου) Αριάδνη Χάγερ (Επ. Καθ. ΓΠΑ) Επταμελής εξεταστική επιτροπή: Aντώνιος Κομινάκης (Αν. Καθ. ΓΠΑ) Ανδρέας Κράνης (Eρευν. B, Παν. Εδιμβούργου) Αριάδνη Χάγερ (Επ. Καθ. ΓΠΑ) Πηνελόπη Μπεμπέλη (Καθ. ΓΠΑ) Δημήτριος Βλαχάκης (Επ. Καθ. ΓΠΑ) Ευάγγελος Ζωίδης (Επ.Καθ. ΓΠΑ) Γεώργιος Θεοδώρου (Επ.Καθ. ΓΠΑ) 2 Εντοπισμός γονιδιωματικών περιοχών και δικτύων γονιδίων που επηρεάζουν παραγωγικές και αναπαραγωγικές ιδιότητες σε πληθυσμούς κρεοπαραγωγικών ορνιθίων Περίληψη Σκοπός της παρούσας διδακτορικής διατριβής ήταν ο εντοπισμός γενετικών δεικτών και υποψηφίων γονιδίων που εμπλέκονται στο γενετικό έλεγχο δύο τυπικών πολυγονιδιακών ιδιοτήτων σε κρεοπαραγωγικά ορνίθια. Μία ιδιότητα σχετίζεται με την ανάπτυξη (σωματικό βάρος στις 35 ημέρες, ΣΒ) και η άλλη με την αναπαραγωγική
    [Show full text]
  • Autocrine IFN Signaling Inducing Profibrotic Fibroblast Responses By
    Downloaded from http://www.jimmunol.org/ by guest on September 23, 2021 Inducing is online at: average * The Journal of Immunology , 11 of which you can access for free at: 2013; 191:2956-2966; Prepublished online 16 from submission to initial decision 4 weeks from acceptance to publication August 2013; doi: 10.4049/jimmunol.1300376 http://www.jimmunol.org/content/191/6/2956 A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Autocrine IFN Signaling Feng Fang, Kohtaro Ooka, Xiaoyong Sun, Ruchi Shah, Swati Bhattacharyya, Jun Wei and John Varga J Immunol cites 49 articles Submit online. Every submission reviewed by practicing scientists ? is published twice each month by Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts http://jimmunol.org/subscription Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html http://www.jimmunol.org/content/suppl/2013/08/20/jimmunol.130037 6.DC1 This article http://www.jimmunol.org/content/191/6/2956.full#ref-list-1 Information about subscribing to The JI No Triage! Fast Publication! Rapid Reviews! 30 days* Why • • • Material References Permissions Email Alerts Subscription Supplementary The Journal of Immunology The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2013 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. This information is current as of September 23, 2021. The Journal of Immunology A Synthetic TLR3 Ligand Mitigates Profibrotic Fibroblast Responses by Inducing Autocrine IFN Signaling Feng Fang,* Kohtaro Ooka,* Xiaoyong Sun,† Ruchi Shah,* Swati Bhattacharyya,* Jun Wei,* and John Varga* Activation of TLR3 by exogenous microbial ligands or endogenous injury-associated ligands leads to production of type I IFN.
    [Show full text]
  • Investigating the Functionality of a Ribosome-Binding Mutant of NAA15
    Varland and Arnesen BMC Res Notes (2018) 11:404 https://doi.org/10.1186/s13104-018-3513-4 BMC Research Notes RESEARCH NOTE Open Access Investigating the functionality of a ribosome‑binding mutant of NAA15 using Saccharomyces cerevisiae Sylvia Varland1,2,3* and Thomas Arnesen1,2,4 Abstract Objective: N-terminal acetylation is a common protein modifcation that occurs preferentially co-translationally as the substrate N-terminus is emerging from the ribosome. The major N-terminal acetyltransferase complex A (NatA) is estimated to N-terminally acetylate more than 40% of the human proteome. To form a functional NatA complex the catalytic subunit NAA10 must bind the auxiliary subunit NAA15, which properly folds NAA10 for correct substrate acetylation as well as anchors the entire complex to the ribosome. Mutations in these two genes are associated with various neurodevelopmental disorders in humans. The aim of this study was to investigate the in vivo functionality of a Schizosaccharomyces pombe NAA15 mutant that is known to prevent NatA from associating with ribosomes, but retains NatA-specifc activity in vitro. Results: Here, we show that Schizosaccharomyces pombe NatA can functionally replace Saccharomyces cerevisiae NatA. We further demonstrate that the NatA ribosome-binding mutant Naa15 ΔN K6E is unable to rescue the temper- ature-sensitive growth phenotype of budding yeast lacking NatA. This fnding indicates the in vivo importance of the co-translational nature of NatA-mediated N-terminal acetylation. Keywords: N-terminal acetyltransferase, NatA, NAA10, NAA15, N-terminal acetylation, Ribosome association, Saccharomyces cerevisiae Introduction acetyltransferases (NATs). Te biological importance of N-terminal acetylation is one of the most common this protein modifcation is underscored by the fact that protein modifcations in eukaryotes, occurring on dysfunctional NATs are implicated in a variety of devel- approximately 80–90% of all human proteins [1–3].
    [Show full text]
  • Supplementary Information
    SUPPLEMENTARY INFORMATION 1. SUPPLEMENTARY FIGURE LEGENDS Supplementary Figure 1. Long-term exposure to sorafenib increases the expression of progenitor cell-like features. A) mRNA expression levels of PROM-1 (CD133), THY-1 (CD90), EpCAM, KRT19, and VIM assessed by quantitative real-time PCR. Data represent the mean expression value for a gene in each phenotypic type of cells, displayed as fold-changes normalized to 1 (expression value of its corresponding parental non-treated cell line). Expression level is relative to the GAPDH gene. Bars indicate standard deviation. Significant statistical differences are set at p<0.05. B) Immunocitochemical staining of CD90 and vimentin in Hep3B sorafenib resistant cell line and its parental cell line. C) Western blot analysis comparing protein levels in resistant Hu6 and Hep3B cells vs their corresponding parental cells lines. Supplementary Figure 2. Efficacy of gene silencing of IGF1R and FGFR1 and evaluation of MAPK14 signaling activation. IGF1R and FGFR1 knockdown expression 48h after transient transfection with siRNAs (50 nM), in non-treated parental cells and sorafenib-acquired resistant tumor derived cells was assessed by quantitative RT-PCR (A) and western blot (B). C) Activation status of MAPK14 signaling was evaluated by western blot analysis in vivo, in tumors with acquired resistance to sorafenib in comparison to non-treated tumors (right panel), as well as in in vitro, in sorafenib resistant cell lines vs parental non-treated. Supplementary Figure 3. Gene expression levels of several pro-angiogenic factors. mRNA expression levels of FGF1, FGF2, VEGFA, IL8, ANGPT2, KDR, FGFR3, FGFR4 assessed by quantitative real-time PCR in tumors harvested from mice.
    [Show full text]
  • Prospective Lncrna-Mirna-Mrna Regulatory
    INTERNATIONAL JOURNAL OF MOLECULAR MEDICINE 40: 1895-1906, 2017 Prospective lncRNA-miRNA-mRNA regulatory network of long non-coding RNA LINC00968 in non-small cell lung cancer A549 cells: A miRNA microarray and bioinformatics investigation DONG-YAO LI1*, WEN-JIE CHEN1*, LEI LUO1, YONG-KUN WANG1, JUN SHANG1, YU ZHANG2, GANG CHEN2 and SHI-KANG LI1 Departments of 1Thoracic and Cardiovascular Surgery and 2Pathology, The First Affiliated Hospital of Guangxi Medical University, Nanning, Guangxi 530021, P.R. China Received December 12, 2016; Accepted October 5, 2017 DOI: 10.3892/ijmm.2017.3187 Abstract. Accumulating evidence suggests that the dysregula- adhesion. An lncRNA-miRNA-mRNA regulatory network and tion of long non-coding RNAs (lncRNAs) serves vital roles a protein-protein interaction network were then constructed. in the incidence and progression of lung cancer. However, the Eventually, a prospective lncRNA-miRNA-mRNA regulatory molecular mechanisms of LINC00968, a recently identified network of LINC00968, three miRNAs (miR-9, miR-22 and lncRNA, remain unknown. The objective of present study was miR-4536) and two genes (polo-like kinase 1 and exportin-1) to investigate the role of a prospective lncRNA-miRNA-mRNA was obtained following validation in the Cancer Genome Atlas network regulated by LINC00968 in non-small cell lung database. These results may provide novel insights to support cancer cells. Following the transfection of lentiviruses carrying future research into lncRNA in lung cancer. LINC00968 into A549 cells, the microRNA (miRNA) expres- sion profile of the cells in response to the overexpression of Introduction LINC00968 was detected using an miRNA microarray. Five differentially expressed miRNAs (DEMs) with LINC00968 Lung cancer results in the death of ~1.59 million indi- overexpression were obtained, including miR-9-3p, miR-22-5p, viduals worldwide each year, and is a leading cause of cancer miR-668-3p, miR-3675-3p and miR-4536-3p.
    [Show full text]