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Mouse Slc22a14 Knockout Project (CRISPR/Cas9)

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https://www.alphaknockout.com Mouse Slc22a14 Knockout Project (CRISPR/Cas9) Objective: To create a Slc22a14 knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Slc22a14 gene (NCBI Reference Sequence: NM_001037749 ; Ensembl: ENSMUSG00000070280 ) is located on Mouse chromosome 9. 12 exons are identified, with the ATG start codon in exon 3 and the TAG stop codon in exon 12 (Transcript: ENSMUST00000093775). Exon 3~10 will be selected as target site. Cas9 and gRNA will be co-injected into fertilized eggs for KO Mouse production. The pups will be genotyped by PCR followed by sequencing analysis. Note: Mice homozygous for a knock-out allele exhibit severe male infertility associated with asthenozoospermia, impaired sperm capacitation, decreased fertilization frequency, abnormal sperm flagellar bending, and abnormal sperm annulus morphology. Exon 3 starts from the coding region. Exon 3~10 covers 78.64% of the coding region. The size of effective KO region: ~8985 bp. The KO region does not have any other known gene. Page 1 of 9 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 5 6 7 8 9 10 12 Legends Exon of mouse Slc22a14 Knockout region Page 2 of 9 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 2000 bp section upstream of Exon 3 is aligned with itself to determine if there are tandem repeats. Tandem repeats are found in the dot plot matrix. The gRNA site is selected outside of these tandem repeats. Overview of the Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 1403 bp section downstream of Exon 10 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. Page 3 of 9 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(2000bp) | A(21.15% 423) | C(26.3% 526) | T(32.1% 642) | G(20.45% 409) Note: The 2000 bp section upstream of Exon 3 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Overview of the GC Content Distribution (down) Window size: 300 bp Sequence 12 Summary: Full Length(1403bp) | A(26.44% 371) | C(23.38% 328) | T(26.59% 373) | G(23.59% 331) Note: The 1403 bp section downstream of Exon 10 is analyzed to determine the GC content. No significant high GC-content region is found. So this region is suitable for PCR screening or sequencing analysis. Page 4 of 9 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN -------------------------------------------------------------------------------------------------------------- browser details YourSeq 2000 1 2000 2000 100.0% chr9 - 119181028 119183027 2000 browser details YourSeq 159 804 1373 2000 86.1% chr8 + 70625906 70626325 420 browser details YourSeq 144 811 978 2000 92.2% chr12 - 77150656 77150822 167 browser details YourSeq 144 806 973 2000 91.0% chr1 - 190963661 190963826 166 browser details YourSeq 144 792 964 2000 94.5% chr1 - 52478945 52479119 175 browser details YourSeq 143 692 973 2000 93.9% chr4 - 134694535 134695019 485 browser details YourSeq 142 808 974 2000 91.5% chr4 - 48794236 48794389 154 browser details YourSeq 140 811 964 2000 96.2% chr2 - 69802560 69802728 169 browser details YourSeq 138 808 975 2000 91.8% chr9 - 42529570 42529735 166 browser details YourSeq 138 804 975 2000 90.8% chr13 - 105191691 105191860 170 browser details YourSeq 137 779 976 2000 85.9% chr6 - 79593951 79594116 166 browser details YourSeq 137 811 972 2000 91.1% chr14 - 59580876 59581034 159 browser details YourSeq 137 811 974 2000 94.8% chrX + 74441554 74441717 164 browser details YourSeq 136 799 963 2000 92.1% chr15 - 91450376 91450541 166 browser details YourSeq 136 791 972 2000 96.6% chr13 - 26619361 26619854 494 browser details YourSeq 136 811 971 2000 94.8% chr5 + 93395540 93395700 161 browser details YourSeq 135 807 973 2000 89.5% chr8 - 123099695 123099848 154 browser details YourSeq 135 811 973 2000 89.2% chr1 - 156716450 156716605 156 browser details YourSeq 134 804 961 2000 90.1% chr2 - 162007445 162007595 151 browser details YourSeq 134 811 973 2000 91.1% chr13 - 55605036 55605196 161 Note: The 2000 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 1403 1 1403 1403 100.0% chr9 - 119170640 119172042 1403 browser details YourSeq 98 751 1044 1403 80.4% chr3 - 33688997 33689302 306 browser details YourSeq 95 742 997 1403 85.9% chr12 + 8225855 8226114 260 browser details YourSeq 94 705 1034 1403 79.3% chr13 + 55045860 55046182 323 browser details YourSeq 88 729 1070 1403 88.6% chr18 - 68030823 68031195 373 browser details YourSeq 86 722 864 1403 82.8% chr6 - 115832106 115832239 134 browser details YourSeq 84 701 865 1403 92.0% chr4 - 48745885 48746049 165 browser details YourSeq 83 734 987 1403 91.9% chr6 - 67424962 67425223 262 browser details YourSeq 83 697 858 1403 92.1% chr12 - 25790774 25790935 162 browser details YourSeq 82 700 951 1403 92.8% chr17 + 48783606 48783880 275 browser details YourSeq 81 734 949 1403 89.6% chr6 - 94013648 94013861 214 browser details YourSeq 81 697 876 1403 79.3% chr12 - 17556264 17556432 169 browser details YourSeq 81 732 952 1403 89.3% chr18 + 60542839 60543056 218 browser details YourSeq 81 704 959 1403 91.8% chr1 + 85609011 85706189 97179 browser details YourSeq 79 790 1013 1403 90.0% chr17 - 6418145 6418386 242 browser details YourSeq 79 789 1013 1403 89.3% chr17 + 6667660 6667902 243 browser details YourSeq 79 697 960 1403 86.2% chr1 + 6390296 6390553 258 browser details YourSeq 77 710 864 1403 86.7% chr12 - 11066489 11066669 181 browser details YourSeq 76 722 864 1403 88.1% chr2 - 118243558 118243698 141 browser details YourSeq 76 711 986 1403 87.8% chr7 + 63518334 63518614 281 Note: The 1403 bp section downstream of Exon 10 is BLAT searched against the genome. No significant similarity is found. Page 5 of 9 https://www.alphaknockout.com Gene and protein information: Slc22a14 solute carrier family 22 (organic cation transporter), member 14 [ Mus musculus (house mouse) ] Gene ID: 382113, updated on 3-Sep-2019 Gene summary Official Symbol Slc22a14 provided by MGI Official Full Name solute carrier family 22 (organic cation transporter), member 14 provided by MGI Primary source MGI:MGI:2685974 See related Ensembl:ENSMUSG00000070280 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 Gm1128 Expression Restricted expression toward testis adult (RPKM 114.3) See more Orthologs human all Genomic context Location: 9; 9 F3 See Slc22a14 in Genome Data Viewer Exon count: 13 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 9 NC_000075.6 (119169453..119190430, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 9 NC_000075.5 (119078574..119099511, complement) Chromosome 9 - NC_000075.6 Page 6 of 9 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Slc22a14 ENSMUSG00000070280 Description solute carrier family 22 (organic cation transporter), member 14 [Source:MGI Symbol;Acc:MGI:2685974] Gene Synonyms LOC382113 Location Chromosome 9: 119,169,455-119,365,553 reverse strand. GRCm38:CM001002.2 About this gene This gene has 4 transcripts (splice variants), 445 orthologues, 26 paralogues and is a member of 1 Ensembl protein family. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Slc22a14-201 ENSMUST00000093775.11 2213 629aa ENSMUSP00000091289.5 Protein coding CCDS23609 Q497L9 TSL:1 GENCODE basic APPRIS P1 Slc22a14-204 ENSMUST00000170400.8 2110 629aa ENSMUSP00000131982.2 Protein coding CCDS23609 Q497L9 TSL:5 GENCODE basic APPRIS P1 Slc22a14-203 ENSMUST00000152061.1 589 196aa ENSMUSP00000117967.1 Protein coding - F7AMC9 CDS 5' and 3' incomplete TSL:3 Slc22a14-202 ENSMUST00000127794.1 340 20aa ENSMUSP00000120144.1 Protein coding - D3YUH1 CDS 3' incomplete TSL:5 Page 7 of 9 https://www.alphaknockout.com 216.10 kb Forward strand 119.20Mb 119.25Mb 119.30Mb 119.35Mb Genes Gm47289-201 >lncRNA Acaa1a-208 >nonsense mediated decay (Comprehensive set... Gm10608-201 >processed pseudogene Xylb-204 >nonsense mediated decay Xylb-205 >nonsense mediated decay Acaa1a-206 >protein coding Acaa1a-210 >protein coding Acaa1a-201 >protein coding Acaa1a-209 >retained intron Acaa1a-204 >retained intron Acaa1a-207 >protein coding Acaa1a-203 >protein coding Acaa1a-205 >retained intron Acaa1a-202 >retained intron Xylb-201 >protein coding Xylb-203 >lncRNA Contigs < AC055818.9 < AC128702.4 Genes (Comprehensive set... < Slc22a14-201protein coding < Slc22a13b-201polymorphic pseudogene < Gm22729-201snoRNA< Myd88-201protein coding < Slc22a14-204protein coding < Slc22a14-203protein coding < Oxsr1-201protein coding < Myd88-203retained intron < Slc22a14-202protein coding < Oxsr1-202nonsense mediated
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  • 82712428.Pdf

    82712428.Pdf

    View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector Available online at www.sciencedirect.com Genomics 90 (2007) 595–609 www.elsevier.com/locate/ygeno Identification of six putative human transporters with structural similarity to the drug transporter SLC22 family ⁎ Josefin A. Jacobsson, Tatjana Haitina, Jonas Lindblom, Robert Fredriksson Department of Neuroscience, Unit of Pharmacology, Uppsala University, BMC, Uppsala SE 75124, Sweden Received 5 February 2007; accepted 24 March 2007 Available online 22 August 2007 Abstract The solute carrier family 22 (SLC22) is a large family of organic cation and anion transporters. These are transmembrane proteins expressed predominantly in kidneys and liver and mediate the uptake and excretion of environmental toxins, endogenous substances, and drugs from the body. Through a comprehensive database search we identified six human proteins not yet cloned or annotated in the reference sequence databases. Five of these belong to the SLC22 family, SLC22A20, SLC22A23, SLC22A24, SLC22A25, and SPNS3, and the sixth gene, SVOPL, is a paralog to the synaptic vesicle protein SVOP. We identified the orthologs for these genes in mouse and rat and additional homologous proteins and performed the first phylogenetic analysis on the entire SLC22 family in human, mouse, and rat. In addition, we performed a phylogenetic analysis which showed that SVOP and SV2A-C are, in a comparison with all vertebrate proteins, most similar to the SLC22 family. Finally, we performed a tissue localization study on 15 genes on a panel of 30 rat tissues using quantitative real-time polymerase chain reaction.
  • Supplemental Figures 04 12 2017

    Supplemental Figures 04 12 2017

    Jung et al. 1 SUPPLEMENTAL FIGURES 2 3 Supplemental Figure 1. Clinical relevance of natural product methyltransferases (NPMTs) in brain disorders. (A) 4 Table summarizing characteristics of 11 NPMTs using data derived from the TCGA GBM and Rembrandt datasets for 5 relative expression levels and survival. In addition, published studies of the 11 NPMTs are summarized. (B) The 1 Jung et al. 6 expression levels of 10 NPMTs in glioblastoma versus non‐tumor brain are displayed in a heatmap, ranked by 7 significance and expression levels. *, p<0.05; **, p<0.01; ***, p<0.001. 8 2 Jung et al. 9 10 Supplemental Figure 2. Anatomical distribution of methyltransferase and metabolic signatures within 11 glioblastomas. The Ivy GAP dataset was downloaded and interrogated by histological structure for NNMT, NAMPT, 12 DNMT mRNA expression and selected gene expression signatures. The results are displayed on a heatmap. The 13 sample size of each histological region as indicated on the figure. 14 3 Jung et al. 15 16 Supplemental Figure 3. Altered expression of nicotinamide and nicotinate metabolism‐related enzymes in 17 glioblastoma. (A) Heatmap (fold change of expression) of whole 25 enzymes in the KEGG nicotinate and 18 nicotinamide metabolism gene set were analyzed in indicated glioblastoma expression datasets with Oncomine. 4 Jung et al. 19 Color bar intensity indicates percentile of fold change in glioblastoma relative to normal brain. (B) Nicotinamide and 20 nicotinate and methionine salvage pathways are displayed with the relative expression levels in glioblastoma 21 specimens in the TCGA GBM dataset indicated. 22 5 Jung et al. 23 24 Supplementary Figure 4.
  • Supplementary Data

    Supplementary Data

    SUPPLEMENTAL INFORMATION A study restricted to chemokine receptors as well as a genome-wide transcript analysis uncovered CXCR4 as preferentially expressed in Ewing's sarcoma (Ewing's sarcoma) cells of metastatic origin (Figure 4). Transcriptome analyses showed that in addition to CXCR4, genes known to support cell motility and invasion topped the list of genes preferentially expressed in metastasis-derived cells (Figure 4D). These included kynurenine 3-monooxygenase (KMO), galectin-1 (LGALS1), gastrin-releasing peptide (GRP), procollagen C-endopeptidase enhancer (PCOLCE), and ephrin receptor B (EPHB3). KMO, a key enzyme of tryptophan catabolism, has not been linked to metastasis. Tryptophan and its catabolites, however, are involved in immune evasion by tumors, a process that can assist in tumor progression and metastasis (1). LGALS1, GRP, PCOLCE and EPHB3 have been linked to tumor progression and metastasis of several cancers (2-4). Top genes preferentially expressed in L-EDCL included genes that suppress cell motility and/or potentiate cell adhesion such as plakophilin 1 (PKP1), neuropeptide Y (NPY), or the metastasis suppressor TXNIP (5-7) (Figure 4D). Overall, L-EDCL were enriched in gene sets geared at optimizing nutrient transport and usage (Figure 4D; Supplementary Table 3), a state that may support the early stages of tumor growth. Once tumor growth outpaces nutrient and oxygen supplies, gene expression programs are usually switched to hypoxic response and neoangiogenesis, which ultimately lead to tumor egress and metastasis. Accordingly, gene sets involved in extracellular matrix remodeling, MAPK signaling, and response to hypoxia were up-regulated in M-EDCL (Figure 4D; Supplementary Table 4), consistent with their association to metastasis in other cancers (8, 9).