DOCSLIB.ORG

Mouse Mapk14 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Mapk14 Conditional Knockout Project (CRISPR/Cas9) http://www.alphaknockout.com/ Mouse Mapk14 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mapk14 conditional knockout mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mapk14 gene ( NCBI Reference Sequence: NM_001168508 ; Ensembl: ENSMUSG00000053436 ) is located on mouse chromosome 17. 12 exons are identified , with the ATG start codon in exon 1 and the TGA stop codon in exon 12 (Transcript: ENSMUST00000004990). Exon 3 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the mouse Mapk14 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-369C13 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 various null mutations are embryonic to perinatal lethal showing multiple organ system defects. Mice homozygous for a knock-out mutation exhibit abnormal myoblast differentiation and delayed myofiber growth and maturation. The knockout of Exon 3 will result in frameshift of the gene, and covers 5.46% of the coding region. The size of intron 2 for 5'-loxP site insertion: 1479 bp, and the size of intron 3 for 3'-loxP site insertion: 9302 bp. The size of effective cKO region: ~1500 bp. This strategy is designed based on genetic information in existing databases. Due to the complexity of biological processes, all risk of loxP insertion on gene transcription, RNA splicing and protein translation cannot be predicted at existing technological level. Page 1 of 8 http://www.alphaknockout.com/ Overview of the Targeting Strategy Wildtype allele gRNA region 5' gRNA region 3' 1 2 3 12 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mapk14 Homology arm cKO region loxP site Page 2 of 8 http://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(7059bp) | A(26.22% 1851) | C(19.99% 1411) | G(25.06% 1769) | T(28.73% 2028) 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 http://www.alphaknockout.com/ BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr17 + 28712134 28715133 3000 browser details YourSeq 149 1407 1584 3000 94.7% chr13 - 62812446 62812635 190 browser details YourSeq 149 1418 1978 3000 84.2% chr5 + 110035337 110035521 185 browser details YourSeq 149 1400 1575 3000 94.5% chr17 + 24145765 24145939 175 browser details YourSeq 146 1412 1590 3000 94.7% chr13 + 79554408 79554592 185 browser details YourSeq 145 1413 1584 3000 95.1% chr3 - 47865335 47865506 172 browser details YourSeq 145 1406 1585 3000 92.2% chr1 - 74553859 74554036 178 browser details YourSeq 144 1411 1584 3000 95.2% chr3 - 93685953 93686132 180 browser details YourSeq 144 1411 1587 3000 94.0% chr15 - 82009757 82009934 178 browser details YourSeq 143 1410 1583 3000 95.1% chr7 - 91454918 91455091 174 browser details YourSeq 143 1410 1580 3000 95.1% chr17 - 19328310 19328484 175 browser details YourSeq 143 1407 1583 3000 90.5% chr1 + 13056804 13056969 166 browser details YourSeq 142 1407 1586 3000 94.0% chr13 + 57677229 57677414 186 browser details YourSeq 142 1410 1583 3000 95.0% chr11 + 68896064 68896297 234 browser details YourSeq 140 1417 1584 3000 95.0% chr3 - 54623284 54623452 169 browser details YourSeq 140 1413 1584 3000 94.4% chr2 - 34905886 34906065 180 browser details YourSeq 140 1412 1582 3000 95.0% chr18 - 66137198 66137611 414 browser details YourSeq 140 1404 1584 3000 90.1% chr17 + 46149465 46149626 162 browser details YourSeq 140 1407 1580 3000 90.1% chr17 + 35128196 35128365 170 browser details YourSeq 140 1405 1584 3000 93.9% chr11 + 84170995 84171191 197 Note: The 3000 bp section upstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. BLAT Search Results (down) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 3000 1 3000 3000 100.0% chr17 + 28715693 28718692 3000 browser details YourSeq 182 1196 1571 3000 86.6% chr1 + 93735934 93736310 377 browser details YourSeq 181 1196 1561 3000 89.6% chr2 - 121909624 121909998 375 browser details YourSeq 175 1196 1571 3000 92.5% chr11 - 119336991 119337475 485 browser details YourSeq 172 1146 1572 3000 86.5% chr11 + 80717484 80717982 499 browser details YourSeq 170 1240 1564 3000 86.9% chr11 - 120934204 120934529 326 browser details YourSeq 170 1141 1540 3000 89.1% chr8 + 23378725 23379131 407 browser details YourSeq 163 1141 1559 3000 90.6% chr5 + 120496420 120496873 454 browser details YourSeq 161 1141 1571 3000 90.6% chr5 + 124850009 124850456 448 browser details YourSeq 159 1138 1564 3000 90.2% chr17 - 12500646 12501109 464 browser details YourSeq 158 1138 1557 3000 90.3% chr3 + 68885304 68885726 423 browser details YourSeq 156 1144 1582 3000 89.6% chr15 - 93114403 93115014 612 browser details YourSeq 154 1141 1562 3000 82.2% chr2 + 12243635 12244039 405 browser details YourSeq 146 1240 1569 3000 87.2% chr7 + 127010545 127010871 327 browser details YourSeq 144 1141 1562 3000 89.9% chr10 - 79635557 79635976 420 browser details YourSeq 143 1196 1564 3000 89.2% chr18 + 48232630 48233056 427 browser details YourSeq 142 1253 1570 3000 86.3% chr10 - 43116765 43117275 511 browser details YourSeq 140 1219 1560 3000 84.4% chr5 - 33937651 33937988 338 browser details YourSeq 139 1240 1571 3000 89.1% chr1 - 172385099 172789427 404329 browser details YourSeq 138 1196 1564 3000 83.7% chr9 + 108453244 108453606 363 Note: The 3000 bp section downstream of Exon 3 is BLAT searched against the genome. No significant similarity is found. Page 4 of 8 http://www.alphaknockout.com/ Gene and protein information: Mapk14 mitogen-activated protein kinase 14 [ Mus musculus (house mouse) ] Gene ID: 26416, updated on 1-Mar-2021 Gene summary Official Symbol Mapk14 provided by MGI Official Full Name mitogen-activated protein kinase 14 provided by MGI Primary source MGI:MGI:1346865 See related Ensembl:ENSMUSG00000053436 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 p3; CSB; Crk; Mxi; p38; Crk1; Mxi2; p38-; p38M; p38a; CSBP2; Csbp1; PRKM14; PRKM15; p38MAPK; p38alpha; p38- Expression alpha Orthologs Ubiquitous expression in spleen adult (RPKM 35.2), thymus adult (RPKM 34.4) and 28 other tissues See more human all NEW Try the new Gene table Try the new Transcript table Genomic context Location: 17 A3.3; 17 14.85 cM See Mapk14 in Genome Data Viewer Exon count: 15 Annotation release Status Assembly Chr Location 109 current GRCm39 (GCF_000001635.27) 17 NC_000083.7 (28910243..28967379) 108.20200622 previous assembly GRCm38.p6 (GCF_000001635.26) 17 NC_000083.6 (28691342..28748405) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 17 NC_000083.5 (28828287..28885350) Chromosome 17 - NC_000083.7 Page 5 of 8 http://www.alphaknockout.com/ Transcript information: This gene has 10 transcripts Gene: Mapk14 ENSMUSG00000053436 Description mitogen-activated protein kinase 14 [Source:MGI Symbol;Acc:MGI:1346865] Gene Synonyms CSBP2, Crk1, Csbp1, Mxi2, p38, p38 MAP Kinase, p38 MAP kinase alpha, p38 MAPK, p38 alpha, p38-alpha, p38MAPK, p38a, p38alpha Location Chromosome 17: 28,910,303-28,967,380 forward strand. GRCm39:CM001010.3 About this gene This gene has 10 transcripts (splice variants), 294 orthologues, 19 paralogues and is associated with 49 phenotypes. Transcripts UniProt Name Transcript ID bp Protein Translation ID Biotype CCDS Flags Match Mapk14- ENSMUST00000114754.8 3592 283aa ENSMUSP00000110402.2 Protein coding CCDS50049 P47811-4 TSL:1 204 GENCODE basic Mapk14- ENSMUST00000004990.14 3562 360aa ENSMUSP00000004990.7 Protein coding CCDS50048 P47811-3 TSL:1 201 GENCODE basic APPRIS ALT1 Mapk14- ENSMUST00000062694.16 3548 360aa ENSMUSP00000061958.9 Protein coding CCDS28583 P47811-1 TSL:1 202 GENCODE basic APPRIS P3 Mapk14- ENSMUST00000114752.3 3148 283aa ENSMUSP00000110400.2 Protein coding CCDS50049 P47811-4 TSL:1 203 GENCODE basic Mapk14- ENSMUST00000114758.9 1478 258aa ENSMUSP00000110406.2 Protein coding - P47811-2 TSL:1 205 GENCODE basic Mapk14- ENSMUST00000233250.2 1066 307aa ENSMUSP00000156692.2 Protein coding - A0A3B2WB60 GENCODE 209 basic Mapk14- ENSMUST00000124886.9 866 227aa ENSMUSP00000116914.3 Protein coding - B2KF34 CDS 3' 206 incomplete TSL:3 Mapk14- ENSMUST00000233811.2 3490 50aa ENSMUSP00000156603.2 Nonsense mediated - A0A3B2WAZ7 - 210 decay Mapk14- ENSMUST00000233095.2 223 No - Processed transcript - - - 208 protein Mapk14- ENSMUST00000151613.2
Load more

Recommended publications
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Molecular Profiling of Peripheral Blood Is Associated with Circulating Tumor Cells Content and Poor Survival in Metastatic Castration-Resistant Prostate Cancer
    www.impactjournals.com/oncotarget/ Oncotarget, Vol. 6, No. 12 Molecular profiling of peripheral blood is associated with circulating tumor cells content and poor survival in metastatic castration-resistant prostate cancer Mercedes Marín-Aguilera1, Òscar Reig1,2, Juan José Lozano3, Natalia Jiménez1, Susana García-Recio1,4, Nadina Erill5, Lydia Gaba2, Andrea Tagliapietra2, Vanesa Ortega2, Gemma Carrera6, Anna Colomer5, Pedro Gascón4 and Begoña Mellado1,2 1 Translational Genomics Group and Targeted Therapeutics in Solid Tumors Group, Institut d’Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain 2 Medical Oncology Department, Hospital Clínic, Barcelona, Spain 3 Bioinformatics Platform Department, Centro de Investigación Biomédica en Red en el Área temática de Enfermedades Hepáticas y Digestivas (CIBEREHD), Hospital Clínic, Barcelona, Spain 4 Laboratory of Translational Oncology, Fundació Clínic per a la Recerca Biomèdica, Barcelona, Spain 5 Althia, Barcelona, Spain 6 Medical Oncology Department, Hospital Plató, Barcelona, Spain Correspondence to: Begoña Mellado, email: [email protected] Keywords: circulating tumor cells, peripheral blood, microarrays, cell search system Received: January 22, 2015 Accepted: February 14, 2015 Published: March 12, 2015 This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. ABSTRACT The enumeration of circulating
    [Show full text]
  • IL21R Expressing CD14+CD16+ Monocytes Expand in Multiple
    Plasma Cell Disorders SUPPLEMENTARY APPENDIX IL21R expressing CD14 +CD16 + monocytes expand in multiple myeloma patients leading to increased osteoclasts Marina Bolzoni, 1 Domenica Ronchetti, 2,3 Paola Storti, 1,4 Gaetano Donofrio, 5 Valentina Marchica, 1,4 Federica Costa, 1 Luca Agnelli, 2,3 Denise Toscani, 1 Rosanna Vescovini, 1 Katia Todoerti, 6 Sabrina Bonomini, 7 Gabriella Sammarelli, 1,7 Andrea Vecchi, 8 Daniela Guasco, 1 Fabrizio Accardi, 1,7 Benedetta Dalla Palma, 1,7 Barbara Gamberi, 9 Carlo Ferrari, 8 Antonino Neri, 2,3 Franco Aversa 1,4,7 and Nicola Giuliani 1,4,7 1Myeloma Unit, Dept. of Medicine and Surgery, University of Parma; 2Dept. of Oncology and Hemato-Oncology, University of Milan; 3Hematology Unit, “Fondazione IRCCS Ca’ Granda”, Ospedale Maggiore Policlinico, Milan; 4CoreLab, University Hospital of Parma; 5Dept. of Medical-Veterinary Science, University of Parma; 6Laboratory of Pre-clinical and Translational Research, IRCCS-CROB, Referral Cancer Center of Basilicata, Rionero in Vulture; 7Hematology and BMT Center, University Hospital of Parma; 8Infectious Disease Unit, University Hospital of Parma and 9“Dip. Oncologico e Tecnologie Avanzate”, IRCCS Arcispedale Santa Maria Nuova, Reggio Emilia, Italy ©2017 Ferrata Storti Foundation. This is an open-access paper. doi:10.3324/haematol. 2016.153841 Received: August 5, 2016. Accepted: December 23, 2016. Pre-published: January 5, 2017. Correspondence: [email protected] SUPPLEMENTAL METHODS Immunophenotype of BM CD14+ in patients with monoclonal gammopathies. Briefly, 100 μl of total BM aspirate was incubated in the dark with anti-human HLA-DR-PE (clone L243; BD), anti-human CD14-PerCP-Cy 5.5, anti-human CD16-PE-Cy7 (clone B73.1; BD) and anti-human CD45-APC-H 7 (clone 2D1; BD) for 20 min.
    [Show full text]
  • Cellular and Molecular Signatures in the Disease Tissue of Early
    Cellular and Molecular Signatures in the Disease Tissue of Early Rheumatoid Arthritis Stratify Clinical Response to csDMARD-Therapy and Predict Radiographic Progression Frances Humby1,* Myles Lewis1,* Nandhini Ramamoorthi2, Jason Hackney3, Michael Barnes1, Michele Bombardieri1, Francesca Setiadi2, Stephen Kelly1, Fabiola Bene1, Maria di Cicco1, Sudeh Riahi1, Vidalba Rocher-Ros1, Nora Ng1, Ilias Lazorou1, Rebecca E. Hands1, Desiree van der Heijde4, Robert Landewé5, Annette van der Helm-van Mil4, Alberto Cauli6, Iain B. McInnes7, Christopher D. Buckley8, Ernest Choy9, Peter Taylor10, Michael J. Townsend2 & Costantino Pitzalis1 1Centre for Experimental Medicine and Rheumatology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, Charterhouse Square, London EC1M 6BQ, UK. Departments of 2Biomarker Discovery OMNI, 3Bioinformatics and Computational Biology, Genentech Research and Early Development, South San Francisco, California 94080 USA 4Department of Rheumatology, Leiden University Medical Center, The Netherlands 5Department of Clinical Immunology & Rheumatology, Amsterdam Rheumatology & Immunology Center, Amsterdam, The Netherlands 6Rheumatology Unit, Department of Medical Sciences, Policlinico of the University of Cagliari, Cagliari, Italy 7Institute of Infection, Immunity and Inflammation, University of Glasgow, Glasgow G12 8TA, UK 8Rheumatology Research Group, Institute of Inflammation and Ageing (IIA), University of Birmingham, Birmingham B15 2WB, UK 9Institute of
    [Show full text]
  • Supplemental Information
    Supplemental information Dissection of the genomic structure of the miR-183/96/182 gene. Previously, we showed that the miR-183/96/182 cluster is an intergenic miRNA cluster, located in a ~60-kb interval between the genes encoding nuclear respiratory factor-1 (Nrf1) and ubiquitin-conjugating enzyme E2H (Ube2h) on mouse chr6qA3.3 (1). To start to uncover the genomic structure of the miR- 183/96/182 gene, we first studied genomic features around miR-183/96/182 in the UCSC genome browser (http://genome.UCSC.edu/), and identified two CpG islands 3.4-6.5 kb 5’ of pre-miR-183, the most 5’ miRNA of the cluster (Fig. 1A; Fig. S1 and Seq. S1). A cDNA clone, AK044220, located at 3.2-4.6 kb 5’ to pre-miR-183, encompasses the second CpG island (Fig. 1A; Fig. S1). We hypothesized that this cDNA clone was derived from 5’ exon(s) of the primary transcript of the miR-183/96/182 gene, as CpG islands are often associated with promoters (2). Supporting this hypothesis, multiple expressed sequences detected by gene-trap clones, including clone D016D06 (3, 4), were co-localized with the cDNA clone AK044220 (Fig. 1A; Fig. S1). Clone D016D06, deposited by the German GeneTrap Consortium (GGTC) (http://tikus.gsf.de) (3, 4), was derived from insertion of a retroviral construct, rFlpROSAβgeo in 129S2 ES cells (Fig. 1A and C). The rFlpROSAβgeo construct carries a promoterless reporter gene, the β−geo cassette - an in-frame fusion of the β-galactosidase and neomycin resistance (Neor) gene (5), with a splicing acceptor (SA) immediately upstream, and a polyA signal downstream of the β−geo cassette (Fig.
    [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]
  • Novel Targets of Apparently Idiopathic Male Infertility
    International Journal of Molecular Sciences Review Molecular Biology of Spermatogenesis: Novel Targets of Apparently Idiopathic Male Infertility Rossella Cannarella * , Rosita A. Condorelli , Laura M. Mongioì, Sandro La Vignera * and Aldo E. Calogero Department of Clinical and Experimental Medicine, University of Catania, 95123 Catania, Italy; [email protected] (R.A.C.); [email protected] (L.M.M.); [email protected] (A.E.C.) * Correspondence: [email protected] (R.C.); [email protected] (S.L.V.) Received: 8 February 2020; Accepted: 2 March 2020; Published: 3 March 2020 Abstract: Male infertility affects half of infertile couples and, currently, a relevant percentage of cases of male infertility is considered as idiopathic. Although the male contribution to human fertilization has traditionally been restricted to sperm DNA, current evidence suggest that a relevant number of sperm transcripts and proteins are involved in acrosome reactions, sperm-oocyte fusion and, once released into the oocyte, embryo growth and development. The aim of this review is to provide updated and comprehensive insight into the molecular biology of spermatogenesis, including evidence on spermatogenetic failure and underlining the role of the sperm-carried molecular factors involved in oocyte fertilization and embryo growth. This represents the first step in the identification of new possible diagnostic and, possibly, therapeutic markers in the field of apparently idiopathic male infertility. Keywords: spermatogenetic failure; embryo growth; male infertility; spermatogenesis; recurrent pregnancy loss; sperm proteome; DNA fragmentation; sperm transcriptome 1. Introduction Infertility is a widespread condition in industrialized countries, affecting up to 15% of couples of childbearing age [1]. It is defined as the inability to achieve conception after 1–2 years of unprotected sexual intercourse [2].
    [Show full text]
  • Supporting Information
    Supporting Information Table S1. List of confirmed SLC transporters represented in Canine GeneChip. SLC family Members detected Members not detected SLC1: The high affinity glutamate and neutral amino acid SLC1A1 SLC1A2, SLC1A3, SLC1A6 transporter family SLC2: The facilitative GLUT transporter family SLC2A1, SLC2A8 SLC2A3, SLC2A9 SLC3: The heavy subunits of the heteromeric amino acid SLC3A1 transporters SLC4: The bicarbonate transporter family SLC4A11 SLC4A4, SLC4A8 SLC5: The sodium glucose cotransporter family SLC5A6 SLC5A3, SLC5A10, SLC5A12 SLC6: The sodium- and chloride- dependent SLC6A6, SLC6A12 SLCA18 neurotransmitter transporter family SLC7: The cationic amino acid transporter/glycoprotein- NR associated family SLC8: The Na+/Ca2+ exchanger family SLC8A1 SLC9: The Na+/H+ exchanger family SLC9A1, SLC9A6, SLC9A9 SLC10: The sodium bile salt cotransport family SLC10A2 SLC11: The proton coupled metal ion transporter family NR SLC12: The electroneutral cation-Cl cotransporter family SLC12A3, SLC12A6, SLC12A8 SLC13: The human Na+-sulfate/carboxylate cotransporter SLC13A2 family SLC14: The urea transporter family NR SLC15: The proton oligopeptide cotransporter family SLC15A2, SLC15A4 SLC15A1 SLC16: The monocarboxylate transporter family SLC16A13 SLC16A4 SLC17: The vesicular glutamate transporter family SLC17A3, SLC17A7 SLC18: The vesicular amine transporter family NR SLC19: The folate/thiamine transporter family NR SLC20: The type III Na+-phosphate cotransporter family NR SLC21/SLCO: The organic anion transporting family SLC21A3, SLC21A8,
    [Show full text]
  • Improved De Novo Genome Assembly: Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost
    bioRxiv preprint doi: https://doi.org/10.1101/128348; this version posted April 19, 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. Improved de novo Genome Assembly: Linked-Read Sequencing Combined with Optical Mapping Produce a High Quality Mammalian Genome at Relatively Low Cost Mohr DW1*, Naguib A2, Weisenfeld NI3, Kumar V3, Shah P3, Church DM3, Jaffe D3 and AF Scott1* 1Genetic Resources Core Facility, McKusick-Nathans Institute of Genetic Medicine, Johns Hopkins University School of Medicine, Baltimore, MD 21287 2BioNano Genomics, Inc., 9640 ToWne Centre Dr, Suite 100, San Diego, CA 92121 310X Genomics, Inc., 7068 Koll Center ParkWay #401, Pleasanton, CA 94566 Abstract: Current short-read methods have come to dominate genome sequencing because they are cost-effective, rapid, and accurate. HoWever, short reads are most applicable when data can be aligned to a known reference. Two new methods for de novo assembly are linked-reads and restriction-site labeled optical maps. We combined commercial applications of these technologies for genome assembly of an endangered mammal, the HaWaiian Monk seal. We show that the linked-reads produced With 10X Genomics Chromium chemistry and assembled With Supernova v1.1 software produced scaffolds with an N50 of 22.23 Mbp with the longest individual scaffold of 84.06 Mbp. When combined With Bionano Genomics optical maps using Bionano RefAligner, the scaffold N50 increased to 29.65 Mbp for a total of 170 hybrid scaffolds, the longest of which was 84.78 Mbp.
    [Show full text]
  • REVIEW Drug Transporters, the Blood–Testis Barrier, and Spermatogenesis
    207 REVIEW Drug transporters, the blood–testis barrier, and spermatogenesis Linlin Su, Dolores D Mruk and C Yan Cheng The Mary M Wohlford Laboratory for Male Contraceptive Research, Center for Biomedical Research, Population Council, 1230 York Avenue, New York, New York 10065, USA (Correspondence should be addressed to C Y Cheng; Email: [email protected]) Abstract The blood–testis barrier (BTB), which is created by adjacent elongating spermatids, and elongated spermatids, suggesting Sertoli cells near the basement membrane, serves as a that the developing germ cells are also able to selectively pump ‘gatekeeper’ to prohibit harmful substances from reaching drugs ‘in’ and/or ‘out’ via influx or efflux pumps. We review developing germ cells, most notably postmeiotic spermatids. herein the latest developments regarding the role of drug The BTB also divides the seminiferous epithelium into the transporters in spermatogenesis. We also propose a model basal and adluminal (apical) compartment so that postmeiotic utilized by the testis to protect germ cell development from spermatid development, namely spermiogenesis, can take place ‘harmful’ environmental toxicants and xenobiotics and/or in a specialized microenvironment in the apical compartment from ‘therapeutic’ substances (e.g. anticancer drugs). We also behind the BTB. The BTB also contributes, at least in part, to discuss how drug transporters that are supposed to protect the immune privilege status of the testis, so that anti-sperm spermatogenesis can work against the testis in some instances. antibodies are not developed against antigens that are expressed For example, when drugs (e.g. male contraceptives) that can transiently during spermatogenesis. Recent studies have shown perturb germ cell adhesion and/or maturation are actively that numerous drug transporters are expressed by Sertoli cells.
    [Show full text]
  • The Molecular Characterization and Expression of New Human SLC26 Anion Transporters Minna Kujala-Myllynen
    Helsinki University Biomedical Dissertations No. 69 The Molecular Characterization and Expression of New Human SLC26 Anion Transporters Minna Kujala-Myllynen Department of Medical Genetics University of Helsinki Finland Academic dissertation To be publicly discussed with the permission of the Faculty of Medicine, University of Helsinki, in the large lecture hall of Haartman Institute, Haartmaninkatu 3, Helsinki, on November 18th 2005, at 12 noon. Supervised by: Juha Kere, Professor Department of Biosciences at Novum Karolinska Institute Stockholm, Sweden and Department of Medical Genetics University of Helsinki Helsinki, Finland Reviewed by: Hannu Jalanko, MD, PhD, Adjunct Professor Hospital for Children and Adolescents University of Helsinki Helsinki, Finland and Anne Räisänen-Sokolowski, MD, PhD, Adjunct Professor Department of Pathology University of Helsinki, and Helsinki University Central Hospital Helsinki, Finland Official opponent: Per-Henrik Groop, MD, DMSc, Adjunct Professor Folkhälsan Research Center University of Helsinki Helsinki, Finland ISSN 1457-8433 ISBN 952-10-2735-5 (paperback) ISBN 952-10-2736-3 (PDF) Yliopistopaino Helsinki 2005 To My Family Table of Contents List of Original Publications ................................................................................. 13 Abstract ................................................................................................................. 16 Introduction ..........................................................................................................
    [Show full text]
  • SLC26A8 (N-15): Sc-169329
    SAN TA C RUZ BI OTEC HNOL OG Y, INC . SLC26A8 (N-15): sc-169329 BACKGROUND PRODUCT SLC26A8 (solute carrier family 26, member 8) is a 970 amino acid multi-pass Each vial contains 200 µg IgG in 1.0 ml of PBS with < 0.1% sodium azide membrane protein that contains 14 putative transmembrane regions with cyto - and 0.1% gelatin. plasmic N- and C-termini. SLC26A8 shares 26% identity with pendrin, 28% Blocking peptide available for competition studies, sc-169329 P, (100 µg identity with DRA, and interacts with RACGAP1. SLC26A8 acts as a DIDS- peptide in 0.5 ml PBS containing < 0.1% sodium azide and 0.2% BSA). sensitive anion exchanger mediating chloride, sulfate and oxalate transport. It has been suggested that SLC26A8 fulfills critical anion exchange functions in APPLICATIONS male germ line during meiosis and hence may play a role in spermatogene sis. SLC26A8 may also be involved in a new regulatory pathway linking sulfate SLC26A8 (N-15) is recommended for detection of SLC26A8 of mouse, rat and transport to RhoGTPase signaling in male germ cells. As a critical compo nent human origin by Western Blotting (starting dilution 1:200, dilution range of the sperm annulus, SLC26A8 is essential for correct sperm tail differentia - 1:100-1:1000), immunofluorescence (starting dilution 1:50, dilution range tion and motility and hence male fertility. Existing as four alternatively spliced 1:50-1:500) and solid phase ELISA (starting dilution 1:30, dilution range isoforms, the SLC26A8 gene is conserved in chimpanzee, canine, bovine, 1:30- 1:3000); non cross-reactive with other SLC26A family members.
    [Show full text]