DOCSLIB.ORG

Mouse Abca5 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Abca5 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Abca5 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Abca5 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Abca5 gene (NCBI Reference Sequence: NM_147219 ; Ensembl: ENSMUSG00000018800 ) is located on Mouse chromosome 11. 39 exons are identified, with the ATG start codon in exon 2 and the TGA stop codon in exon 39 (Transcript: ENSMUST00000043961). Exon 2~3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Abca5 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-198N23 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 a knock-out allele exhibit exophthalmos, tremors and collapse of the thyroid gland, and develop a dilated cardiomyopathy with large thrombi due to depression of the cardiac function. Severe edema, liver injury and premature death appear to be sensitive to genetic background. Exon 2 starts from about 0.04% of the coding region. The knockout of Exon 2~3 will result in frameshift of the gene. The size of intron 1 for 5'-loxP site insertion: 8228 bp, and the size of intron 3 for 3'-loxP site insertion: 1010 bp. The size of effective cKO region: ~2007 bp. The cKO region does not have any other known gene. Page 1 of 7 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 2 3 4 39 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Abca5 Homology arm cKO region loxP site Page 2 of 7 https://www.alphaknockout.com Overview of the Dot Plot Window size: 10 bp Forward Reverse Complement Sequence 12 Note: The sequence of homologous arms and cKO region is aligned with itself to determine if there are tandem repeats. No significant tandem repeat is found in the dot plot matrix. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution Window size: 300 bp Sequence 12 Summary: Full Length(8507bp) | A(27.02% 2299) | C(18.94% 1611) | T(33.7% 2867) | G(20.34% 1730) Note: The sequence of homologous arms and cKO region is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 3 of 7 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% chr11 - 110329489 110332488 3000 browser details YourSeq 142 2755 2911 3000 95.6% chr5 - 130085150 130085307 158 browser details YourSeq 140 2755 2910 3000 94.1% chr7 - 65872873 65873026 154 browser details YourSeq 139 2750 2911 3000 93.2% chr4 - 109062952 109063113 162 browser details YourSeq 139 2755 2906 3000 96.1% chr1 + 6189653 6189806 154 browser details YourSeq 138 2755 2925 3000 91.3% chr10 + 5624043 5624211 169 browser details YourSeq 137 2701 2903 3000 96.7% chr10 - 107055727 107056050 324 browser details YourSeq 137 2755 2911 3000 96.7% chr7 + 19680215 19680372 158 browser details YourSeq 137 2758 2928 3000 93.1% chr2 + 68973390 68973565 176 browser details YourSeq 137 2755 2910 3000 94.3% chr12 + 86798606 86798763 158 browser details YourSeq 137 2756 2911 3000 95.5% chr1 + 34796871 34797027 157 browser details YourSeq 136 2755 2907 3000 94.8% chr8 - 22873058 22873212 155 browser details YourSeq 136 2757 2910 3000 94.8% chr6 - 113308949 113309102 154 browser details YourSeq 136 2755 2910 3000 92.8% chr12 + 3752958 3753111 154 browser details YourSeq 135 2755 2911 3000 94.2% chr3 - 86619070 86619226 157 browser details YourSeq 135 2755 2910 3000 94.2% chr2 - 181858810 181858966 157 browser details YourSeq 135 2758 2910 3000 94.8% chr14 + 64968420 64968586 167 browser details YourSeq 134 2755 2911 3000 93.0% chr9 - 113804864 113805022 159 browser details YourSeq 134 2747 2906 3000 92.5% chr2 - 84732926 84733085 160 browser details YourSeq 134 2755 2910 3000 92.1% chr17 - 71368834 71368987 154 Note: The 3000 bp section upstream of Exon 2 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr11 - 110324482 110327481 3000 browser details YourSeq 216 2599 2988 3000 89.3% chr2 + 153894020 153894418 399 browser details YourSeq 194 2599 2972 3000 87.3% chr12 + 30764350 30764724 375 browser details YourSeq 190 2685 2993 3000 89.6% chr3 - 129344024 129344335 312 browser details YourSeq 190 2599 2985 3000 91.4% chr2 - 159138552 159138973 422 browser details YourSeq 189 2599 2988 3000 86.0% chr6 - 30892367 30892769 403 browser details YourSeq 185 2637 2988 3000 89.6% chr9 - 107019205 107019555 351 browser details YourSeq 185 2599 2988 3000 91.2% chr2 + 101398708 101399100 393 browser details YourSeq 181 2648 2980 3000 82.7% chr3 - 130613313 130613626 314 browser details YourSeq 181 2721 2985 3000 87.5% chr11 + 77232304 77232561 258 browser details YourSeq 178 2599 2982 3000 88.4% chr11 - 11531984 11532371 388 browser details YourSeq 177 2648 2988 3000 87.7% chr13 - 51235890 51236233 344 browser details YourSeq 177 2648 2988 3000 90.1% chr9 + 115702569 115702914 346 browser details YourSeq 176 2666 2988 3000 86.8% chr4 + 87778370 87778687 318 browser details YourSeq 175 2599 2975 3000 88.9% chr3 - 15121047 15121425 379 browser details YourSeq 175 2704 2996 3000 86.9% chrX + 85159113 85159405 293 browser details YourSeq 174 2648 3000 3000 89.6% chrX - 94442381 94442741 361 browser details YourSeq 174 2648 2988 3000 88.5% chrX + 48335268 48335615 348 browser details YourSeq 172 2599 2967 3000 91.8% chr10 - 22503445 22503835 391 browser details YourSeq 171 2698 2980 3000 87.8% chr14 + 104159010 104159288 279 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Abca5 ATP-binding cassette, sub-family A (ABC1), member 5 [ Mus musculus (house mouse) ] Gene ID: 217265, updated on 12-Aug-2019 Gene summary Official Symbol Abca5 provided by MGI Official Full Name ATP-binding cassette, sub-family A (ABC1), member 5 provided by MGI Primary source MGI:MGI:2386607 See related Ensembl:ENSMUSG00000018800 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 ABC13; mKIAA1888; B930033A02Rik Expression Broad expression in cerebellum adult (RPKM 4.0), cortex adult (RPKM 3.1) and 19 other tissues See more Orthologs human all Genomic context Location: 11; 11 E1 See Abca5 in Genome Data Viewer Exon count: 39 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 11 NC_000077.6 (110269369..110337723, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 11 NC_000077.5 (110130683..110199030, complement) Chromosome 11 - NC_000077.6 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 5 transcripts Gene: Abca5 ENSMUSG00000018800 Description ATP-binding cassette, sub-family A (ABC1), member 5 [Source:MGI Symbol;Acc:MGI:2386607] Gene Synonyms ABC13, B930033A02Rik Location Chromosome 11: 110,269,369-110,337,716 reverse strand. GRCm38:CM001004.2 About this gene This gene has 5 transcripts (splice variants), 215 orthologues, 15 paralogues, is a member of 1 Ensembl protein family and is associated with 13 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Abca5-201 ENSMUST00000043961.11 8231 1642aa ENSMUSP00000047927.5 Protein coding CCDS25591 Q8K448 TSL:1 GENCODE basic APPRIS P1 Abca5-202 ENSMUST00000124714.7 3829 1194aa ENSMUSP00000120708.1 Protein coding - A2AEP5 CDS 3' incomplete TSL:1 Abca5-204 ENSMUST00000134721.1 610 181aa ENSMUSP00000118328.1 Protein coding - A2AEP6 CDS 3' incomplete TSL:5 Abca5-203 ENSMUST00000127318.7 3580 No protein - Retained intron - - TSL:1 Abca5-205 ENSMUST00000148984.1 2684 No protein - lncRNA - - TSL:1 88.35 kb Forward strand 110.26Mb 110.28Mb 110.30Mb 110.32Mb 110.34Mb Contigs < AL603792.12 AL671964.7 > Genes < Abca5-201protein coding (Comprehensive set... < Abca5-202protein coding < Abca5-203retained intron < Abca5-204protein coding < Abca5-205lncRNA Regulatory Build 110.26Mb 110.28Mb 110.30Mb 110.32Mb 110.34Mb Reverse strand 88.35 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 processed transcript Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000043961 < Abca5-201protein coding Reverse strand 68.31 kb ENSMUSP00000047... Transmembrane heli... Low complexity (Seg) Superfamily P-loop containing nucleoside triphosphate hydrolase SMART AAA+ ATPase domain Pfam PF12698 ABC transporter-like PROSITE profiles ABC transporter-like PROSITE patterns ABC transporter, conserved site PANTHER ATP-binding cassette subfamily A member 5 ABC transporter A Gene3D 3.40.50.300 CDD cd03263 All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant splice region variant synonymous variant Scale bar 0 200 400 600 800 1000 1200 1400 1642 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
Load more

Recommended publications
  • Genotyping of Breech Flystrike Resource – Update
    Project No: ON-00515 Contract No: PO4500010753 AWI Project Manager: Bridget Peachey Contractor Name: CSIRO Agriculture and Food Prepared by: Dr Sonja Dominik Publication Date: July 2019 Genotyping of breech flystrike resource – update Published by Australian Wool Innovation Limited, Level 6, 68 Harrington Street, THE ROCKS, NSW, 2000 This publication should only be used as a general aid and is not a substitute for specific advice. To the extent permitted by law, we exclude all liability for loss or damage arising from the use of the information in this publication. AWI invests in research, development, innovation and marketing activities along the global supply chain for Australian wool. AWI is grateful for its funding, which is primarily provided by Australian woolgrowers through a wool levy and by the Australian Government which provides a matching contribution for eligible R&D activities © 2019 Australian Wool Innovation Ltd. All rights reserved. Contents Executive Summary .................................................................................................................... 3 1 Introduction/Hypothesis .................................................................................................... 5 2 Literature Review ............................................................................................................... 6 3 Project Objectives .............................................................................................................. 8 4 Success in Achieving Objectives ........................................................................................
    [Show full text]
  • Genome-Wide Analysis of ATP-Binding
    Tian et al. BMC Genomics (2017) 18:330 DOI 10.1186/s12864-017-3706-6 RESEARCH ARTICLE Open Access Genome-wide analysis of ATP-binding cassette (ABC) transporters in the sweetpotato whitefly, Bemisia tabaci Lixia Tian1, Tianxue Song2, Rongjun He1, Yang Zeng1, Wen Xie1, Qingjun Wu1, Shaoli Wang1, Xuguo Zhou3* and Youjun Zhang1* Abstract Background: ABC transporter superfamily is one of the largest and ubiquitous groups of proteins. Because of their role in detoxification, insect ABC transporters have gained more attention in recent years. In this study, we annotated ABC transporters from a newly sequenced sweetpotato whitefly genome. Bemisia tabaci Q biotype is an emerging global invasive species that has caused extensive damages to field crops as well as ornamental plants. Results: A total of 55 ABC transporters containing all eight described subfamilies (A to H) were identified in the B. tabaci Q genome, including 8 ABCAs, 3 ABCBs, 6 ABCCs, 2 ABCDs, 1 ABCE, 3 ABCFs, 23 ABCGs and 9 ABCHs. In comparison to other species, subfamilies G and H in both phloem- and blood-sucking arthropods are expanded. The temporal expression profiles of these 55 ABC transporters throughout B. tabaci developmental stages and their responses to imidacloprid, a neonicotinoid insecticide, were investigated using RNA-seq analysis. Furthermore, the mRNA expression of 24 ABC transporters (44% of the total) representing all eight subfamilies was confirmed by the quantitative real-time PCR (RT-qPCR). Furthermore, mRNA expression levels estimated by RT-qPCR and RNA-seq analyses were significantly correlated (r =0.684,p <0.01). Conclusions: It is the first genome-wide analysis of the entire repertoire of ABC transporters in B.
    [Show full text]
  • 5' Untranslated Region Elements Show High Abundance and Great
    International Journal of Molecular Sciences Article 0 5 Untranslated Region Elements Show High Abundance and Great Variability in Homologous ABCA Subfamily Genes Pavel Dvorak 1,2,* , Viktor Hlavac 2,3 and Pavel Soucek 2,3 1 Department of Biology, Faculty of Medicine in Pilsen, Charles University, 32300 Pilsen, Czech Republic 2 Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 32300 Pilsen, Czech Republic; [email protected] (V.H.); [email protected] (P.S.) 3 Toxicogenomics Unit, National Institute of Public Health, 100 42 Prague, Czech Republic * Correspondence: [email protected]; Tel.: +420-377593263 Received: 7 October 2020; Accepted: 20 November 2020; Published: 23 November 2020 Abstract: The 12 members of the ABCA subfamily in humans are known for their ability to transport cholesterol and its derivatives, vitamins, and xenobiotics across biomembranes. Several ABCA genes are causatively linked to inborn diseases, and the role in cancer progression and metastasis is studied intensively. The regulation of translation initiation is implicated as the major mechanism in the processes of post-transcriptional modifications determining final protein levels. In the current bioinformatics study, we mapped the features of the 50 untranslated regions (50UTR) known to have the potential to regulate translation, such as the length of 50UTRs, upstream ATG codons, upstream open-reading frames, introns, RNA G-quadruplex-forming sequences, stem loops, and Kozak consensus motifs, in the DNA sequences of all members of the subfamily. Subsequently, the conservation of the features, correlations among them, ribosome profiling data as well as protein levels in normal human tissues were examined. The 50UTRs of ABCA genes contain above-average numbers of upstream ATGs, open-reading frames and introns, as well as conserved ones, and these elements probably play important biological roles in this subfamily, unlike RG4s.
    [Show full text]
  • Transcriptional and Post-Transcriptional Regulation of ATP-Binding Cassette Transporter Expression
    Transcriptional and Post-transcriptional Regulation of ATP-binding Cassette Transporter Expression by Aparna Chhibber DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHILOSOPHY in Pharmaceutical Sciences and Pbarmacogenomies in the Copyright 2014 by Aparna Chhibber ii Acknowledgements First and foremost, I would like to thank my advisor, Dr. Deanna Kroetz. More than just a research advisor, Deanna has clearly made it a priority to guide her students to become better scientists, and I am grateful for the countless hours she has spent editing papers, developing presentations, discussing research, and so much more. I would not have made it this far without her support and guidance. My thesis committee has provided valuable advice through the years. Dr. Nadav Ahituv in particular has been a source of support from my first year in the graduate program as my academic advisor, qualifying exam committee chair, and finally thesis committee member. Dr. Kathy Giacomini graciously stepped in as a member of my thesis committee in my 3rd year, and Dr. Steven Brenner provided valuable input as thesis committee member in my 2nd year. My labmates over the past five years have been incredible colleagues and friends. Dr. Svetlana Markova first welcomed me into the lab and taught me numerous laboratory techniques, and has always been willing to act as a sounding board. Michael Martin has been my partner-in-crime in the lab from the beginning, and has made my days in lab fly by. Dr. Yingmei Lui has made the lab run smoothly, and has always been willing to jump in to help me at a moment’s notice.
    [Show full text]
  • Role and Regulation of the P53-Homolog P73 in the Transformation of Normal Human Fibroblasts
    Role and regulation of the p53-homolog p73 in the transformation of normal human fibroblasts Dissertation zur Erlangung des naturwissenschaftlichen Doktorgrades der Bayerischen Julius-Maximilians-Universität Würzburg vorgelegt von Lars Hofmann aus Aschaffenburg Würzburg 2007 Eingereicht am Mitglieder der Promotionskommission: Vorsitzender: Prof. Dr. Dr. Martin J. Müller Gutachter: Prof. Dr. Michael P. Schön Gutachter : Prof. Dr. Georg Krohne Tag des Promotionskolloquiums: Doktorurkunde ausgehändigt am Erklärung Hiermit erkläre ich, dass ich die vorliegende Arbeit selbständig angefertigt und keine anderen als die angegebenen Hilfsmittel und Quellen verwendet habe. Diese Arbeit wurde weder in gleicher noch in ähnlicher Form in einem anderen Prüfungsverfahren vorgelegt. Ich habe früher, außer den mit dem Zulassungsgesuch urkundlichen Graden, keine weiteren akademischen Grade erworben und zu erwerben gesucht. Würzburg, Lars Hofmann Content SUMMARY ................................................................................................................ IV ZUSAMMENFASSUNG ............................................................................................. V 1. INTRODUCTION ................................................................................................. 1 1.1. Molecular basics of cancer .......................................................................................... 1 1.2. Early research on tumorigenesis ................................................................................. 3 1.3. Developing
    [Show full text]
  • ABCA5 (NM 018672) Human Tagged ORF Clone – RC213865 | Origene
    OriGene Technologies, Inc. 9620 Medical Center Drive, Ste 200 Rockville, MD 20850, US Phone: +1-888-267-4436 [email protected] EU: [email protected] CN: [email protected] Product datasheet for RC213865 ABCA5 (NM_018672) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: ABCA5 (NM_018672) Human Tagged ORF Clone Tag: Myc-DDK Symbol: ABCA5 Synonyms: ABC13; EST90625; HTC3 Vector: pCMV6-Entry (PS100001) E. coli Selection: Kanamycin (25 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RC213865 representing NM_018672 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGTCCACTGCAATTAGGGAGGTAGGAGTTTGGAGACAGACCAGAACACTTCTACTGAAGAATTACTTAA TTAAATGCAGAACCAAAAAGAGTAGTGTTCAGGAAATTCTTTTTCCACTATTTTTTTTATTTTGGTTAAT ATTAATTAGCATGATGCATCCAAATAAGAAATATGAAGAAGTGCCTAATATAGAACTCAATCCTATGGAC AAGTTTACTCTTTCTAATCTAATTCTTGGATATACTCCAGTGACTAATATTACAAGCAGCATCATGCAGA AAGTGTCTACTGATCATCTACCTGATGTCATAATTACTGAAGAATATACAAATGAAAAAGAAATGTTAAC ATCCAGTCTCTCTAAGCCGAGCAACTTTGTAGGTGTGGTTTTCAAAGACTCCATGTCCTATGAACTTCGT TTTTTTCCTGATATGATTCCAGTATCTTCTATTTATATGGATTCAAGAGCTGGCTGTTCAAAATCATGTG AGGCTGCTCAGTACTGGTCCTCAGGTTTCACAGTTTTACAAGCATCCATAGATGCTGCCATTATACAGTT GAAGACCAATGTTTCTCTTTGGAAGGAGCTGGAGTCAACTAAAGCTGTTATTATGGGAGAAACTGCTGTT GTAGAAATAGATACCTTTCCCCGAGGAGTAATTTTAATATACCTAGTTATAGCATTTTCACCTTTTGGAT ACTTTTTGGCAATTCATATCGTAGCAGAAAAAGAAAAAAAAATAAAAGAATTTTTAAAGATAATGGGACT TCATGATACTGCCTTTTGGCTTTCCTGGGTTCTTCTATATACAAGTTTAATTTTTCTTATGTCCCTTCTT ATGGCAGTCATTGCGACAGCTTCTTTGTTATTTCCTCAAAGTAGCAGCATTGTGATATTTCTGCTTTTTT
    [Show full text]
  • Whole-Exome Sequencing Identifies Novel Mutations in ABC Transporter
    Liu et al. BMC Pregnancy and Childbirth (2021) 21:110 https://doi.org/10.1186/s12884-021-03595-x RESEARCH ARTICLE Open Access Whole-exome sequencing identifies novel mutations in ABC transporter genes associated with intrahepatic cholestasis of pregnancy disease: a case-control study Xianxian Liu1,2†, Hua Lai1,3†, Siming Xin1,3, Zengming Li1, Xiaoming Zeng1,3, Liju Nie1,3, Zhengyi Liang1,3, Meiling Wu1,3, Jiusheng Zheng1,3* and Yang Zou1,2* Abstract Background: Intrahepatic cholestasis of pregnancy (ICP) can cause premature delivery and stillbirth. Previous studies have reported that mutations in ABC transporter genes strongly influence the transport of bile salts. However, to date, their effects are still largely elusive. Methods: A whole-exome sequencing (WES) approach was used to detect novel variants. Rare novel exonic variants (minor allele frequencies: MAF < 1%) were analyzed. Three web-available tools, namely, SIFT, Mutation Taster and FATHMM, were used to predict protein damage. Protein structure modeling and comparisons between reference and modified protein structures were performed by SWISS-MODEL and Chimera 1.14rc, respectively. Results: We detected a total of 2953 mutations in 44 ABC family transporter genes. When the MAF of loci was controlled in all databases at less than 0.01, 320 mutations were reserved for further analysis. Among these mutations, 42 were novel. We classified these loci into four groups (the damaging, probably damaging, possibly damaging, and neutral groups) according to the prediction results, of which 7 novel possible pathogenic mutations were identified that were located in known functional genes, including ABCB4 (Trp708Ter, Gly527Glu and Lys386Glu), ABCB11 (Gln1194Ter, Gln605Pro and Leu589Met) and ABCC2 (Ser1342Tyr), in the damaging group.
    [Show full text]
  • Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth
    Mutations in the Cholesterol Transporter Gene ABCA5 Are Associated with Excessive Hair Overgrowth Gina M. DeStefano1, Mazen Kurban2, Kwame Anyane-Yeboa3, Claudia Dall’Armi4,5, Gilbert Di Paolo4,5, Heather Feenstra6, Nanette Silverberg6, Luis Rohena3, Larissa D. Lo´ pez-Cepeda7, Vaidehi Jobanputra4, Katherine A. Fantauzzo2, Maija Kiuru2, Marija Tadin-Strapps1, Antonio Sobrino8, Anna Vitebsky1, Dorothy Warburton1,3, Brynn Levy4, Julio C. Salas-Alanis9, Angela M. Christiano1,2* 1 Department of Genetics and Development, Columbia University, New York, New York, United States of America, 2 Department of Dermatology, Columbia University, New York, New York, United States of America, 3 Department of Pediatrics, Columbia University Medical Center, New York, New York, United States of America, 4 Department of Pathology and Cell Biology, Columbia University, New York, New York, United States of America, 5 Taub Institute for Research on Alzheimer’s Disease and the Aging Brain, Columbia University Medical Center, New York, New York, United States of America, 6 St. Luke’s-Roosevelt Hospital Center, New York, New York, United States of America, 7 Centro Dermatolo´gico Pascua, Mexico City, Mexico, 8 New York Presbyterian Hospital, New York, New York, United States of America, 9 Basic Science, Universidad de Monterrey, Nueva Leon, Mexico Abstract Inherited hypertrichoses are rare syndromes characterized by excessive hair growth that does not result from androgen stimulation, and are often associated with additional congenital abnormalities. In this study, we investigated the genetic defect in a case of autosomal recessive congenital generalized hypertrichosis terminalis (CGHT) (OMIM135400) using whole- exome sequencing. We identified a single base pair substitution in the 59 donor splice site of intron 32 in the ABC lipid transporter gene ABCA5 that leads to aberrant splicing of the transcript and a decrease in protein levels throughout patient hair follicles.
    [Show full text]
  • Role of Genetic Variation in ABC Transporters in Breast Cancer Prognosis and Therapy Response
    International Journal of Molecular Sciences Article Role of Genetic Variation in ABC Transporters in Breast Cancer Prognosis and Therapy Response Viktor Hlaváˇc 1,2 , Radka Václavíková 1,2, Veronika Brynychová 1,2, Renata Koževnikovová 3, Katerina Kopeˇcková 4, David Vrána 5 , Jiˇrí Gatˇek 6 and Pavel Souˇcek 1,2,* 1 Toxicogenomics Unit, National Institute of Public Health, 100 42 Prague, Czech Republic; [email protected] (V.H.); [email protected] (R.V.); [email protected] (V.B.) 2 Biomedical Center, Faculty of Medicine in Pilsen, Charles University, 323 00 Pilsen, Czech Republic 3 Department of Oncosurgery, Medicon Services, 140 00 Prague, Czech Republic; [email protected] 4 Department of Oncology, Second Faculty of Medicine, Charles University and Motol University Hospital, 150 06 Prague, Czech Republic; [email protected] 5 Department of Oncology, Medical School and Teaching Hospital, Palacky University, 779 00 Olomouc, Czech Republic; [email protected] 6 Department of Surgery, EUC Hospital and University of Tomas Bata in Zlin, 760 01 Zlin, Czech Republic; [email protected] * Correspondence: [email protected]; Tel.: +420-267-082-711 Received: 19 November 2020; Accepted: 11 December 2020; Published: 15 December 2020 Abstract: Breast cancer is the most common cancer in women in the world. The role of germline genetic variability in ATP-binding cassette (ABC) transporters in cancer chemoresistance and prognosis still needs to be elucidated. We used next-generation sequencing to assess associations of germline variants in coding and regulatory sequences of all human ABC genes with response of the patients to the neoadjuvant cytotoxic chemotherapy and disease-free survival (n = 105).
    [Show full text]
  • ABCA5 Purified Maxpab Mouse Polyclonal Antibody (B01P)
    ABCA5 purified MaxPab mouse GPNGAGKSTIINILVGDIEPTSGQVFLGDYSSETSEDDD polyclonal antibody (B01P) SLKCMGYCPQINPLWPDTTLQEHFEIYGAVKGMSASD MKEVISRITHALDLKEHLQKTVKKLPAGIKRKLCFALSM Catalog Number: H00023461-B01P LGNPQITLLDEPSTGMDPKAKQHMWRAIRTAFKNRKR AAILTTHYMEEAEAVCDRVAIMVSGQLRCIGTVQHLKS Regulatory Status: For research use only (RUO) KFGKGYFLEIKLKDWIENLEVDRLQREIQYIFPNASRQE SFSSILAYKIPKEDVQSLSQSFFKLEEAKHAFAIEEYSF Product Description: Mouse polyclonal antibody raised SQATLEQVFVELTKEQEEEDNSCGTLNSTLWWERTQ against a full-length human ABCA5 protein. EDRVVF Immunogen: ABCA5 (AAI56257.1, 1 a.a. ~ 1642 a.a) Host: Mouse full-length human protein. Reactivity: Human Sequence: MSTAIREVGVWRQTRTLLLKNYLIKCRTKKSSVQEILFP Applications: WB-Tr LFFLFWLILISMMHPNKKYEEVPNIELNPMDKFTLSNLIL (See our web site product page for detailed applications GYTPVTNITSSIMQKVSTDHLPDVIITEEYTNEKEMLTS information) SLSKPSNFVGVVFKDSMSYELRFFPDMIPVSSIYMDSR AGCSKSCEAAQYWSSGFTVLQASIDAAIIQLKTNVSLW Protocols: See our web site at KELESTKAVIMGETAVVEIDTFPRGVILIYLVIAFSPFGY http://www.abnova.com/support/protocols.asp or product FLAIHIVAEKEKKIKEFLKIMGLHDTAFWLSWVLLYTSLI page for detailed protocols FLMSLLMAVIATASLLFPQSSSIVIFLLFFLYGLSSVFFAL Storage Buffer: In 1x PBS, pH 7.4 MLTPLFKKSKHVGIVEFFVTVAFGFIGLMIILIESFPKSLV WLFSPFCHCTFVIGIAQVMHLEDFNEGASFSNLTAGPY Storage Instruction: Store at -20°C or lower. Aliquot to PLIITIIMLTLNSIFYVLLAVYLDQVIPGEFGLRRSSLYFLK avoid repeated freezing and thawing. PSYWSKSKRNYEELSEGNVNGNISFSEIIEPVSSEFVG KEAIRISGIQKTYRKKGENVEALRNLSFDIYEGQITALLG Entrez GeneID: 23461 HSGTGKSTLMNILCGLCPPSDGFASIYGHRVSEIDEMF
    [Show full text]
  • Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands That Promote Axonal Growth
    Research Article: New Research Development Peripheral Nerve Single-Cell Analysis Identifies Mesenchymal Ligands that Promote Axonal Growth Jeremy S. Toma,1 Konstantina Karamboulas,1,ª Matthew J. Carr,1,2,ª Adelaida Kolaj,1,3 Scott A. Yuzwa,1 Neemat Mahmud,1,3 Mekayla A. Storer,1 David R. Kaplan,1,2,4 and Freda D. Miller1,2,3,4 https://doi.org/10.1523/ENEURO.0066-20.2020 1Program in Neurosciences and Mental Health, Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada, 2Institute of Medical Sciences University of Toronto, Toronto, Ontario M5G 1A8, Canada, 3Department of Physiology, University of Toronto, Toronto, Ontario M5G 1A8, Canada, and 4Department of Molecular Genetics, University of Toronto, Toronto, Ontario M5G 1A8, Canada Abstract Peripheral nerves provide a supportive growth environment for developing and regenerating axons and are es- sential for maintenance and repair of many non-neural tissues. This capacity has largely been ascribed to paracrine factors secreted by nerve-resident Schwann cells. Here, we used single-cell transcriptional profiling to identify ligands made by different injured rodent nerve cell types and have combined this with cell-surface mass spectrometry to computationally model potential paracrine interactions with peripheral neurons. These analyses show that peripheral nerves make many ligands predicted to act on peripheral and CNS neurons, in- cluding known and previously uncharacterized ligands. While Schwann cells are an important ligand source within injured nerves, more than half of the predicted ligands are made by nerve-resident mesenchymal cells, including the endoneurial cells most closely associated with peripheral axons. At least three of these mesen- chymal ligands, ANGPT1, CCL11, and VEGFC, promote growth when locally applied on sympathetic axons.
    [Show full text]
  • The ABCA5 Protein: a Urine Diagnostic Marker for Prostatic Intraepithelial Neoplasia Yo Uji Hu , 1Min Wang,1Karenveverka,2 Fernando U
    Imaging, Diagnosis, Prognosis The ABCA5 Protein: A Urine Diagnostic Marker for Prostatic Intraepithelial Neoplasia Yo uji Hu , 1Min Wang,1KarenVeverka,2 Fernando U. Garcia,1and Mark E. Stearns1 Abstract Purpose: To develop a urine diagnostic test for preneoplastic intraepithelial neoplasia of the prostate. Experimental Design: We have used a DNA-binding assay and electrophoretic mobility shift assays (EMSA) to screen for novelduplexed DNA-binding sequences, which bind protein(s) overexpressed in crude protein extracts from high-grade prostatic intraepithelial neoplasia (HGPIN). EMSAs, immunohistochemistry, and ELISAs were used to measure expression of the ABCA5 protein identified as a specific marker in prostate tissue and patient urine. Results: Following screening of 4,096 sequences, an 8-bp dsDNA sequence (i.e.,TCCAGCGA) was identified, which binds the ABCA5 protein, a member of the ATP-binding cassette multidrug resistant family. EMSAs showed that ABCA5 was overexpressed in HGPIN tissue (n =11/11)and in the urine of patients with HGPIN (n = 18/18) but was not expressed in prostate cancer, benign prostatic hyperplasia, or stroma. Immunohistochemistry indicated that ABCA5 was overex- pressed in foci of intermediate basalcellsin normalglands and in HGPIN. ABCA5 was faintly expressed in prostate cancer glands. ELISAs showed in‘blinded studies’ that ABCA5 was a highly sensitive (>98% sensitivity) urine diagnostic marker for HGPIN in biopsy-positive patients (n = 107) at a ‘cutoff’of 25 ng/mL. ABCA5 was present at very low levels (i.e., <25 ng/mL) in the urine of patients diagnosed with benign prostatic hyperplasia (n = 79) or prostatitis or kidney and bladder cancer (>86% specificity).
    [Show full text]