DOCSLIB.ORG

Mouse Mcf2 Conditional Knockout Project (CRISPR/Cas9)

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Mouse Mcf2 Conditional Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Mcf2 Conditional Knockout Project (CRISPR/Cas9) Objective: To create a Mcf2 conditional knockout Mouse model (C57BL/6J) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Mcf2 gene (NCBI Reference Sequence: NM_001289731 ; Ensembl: ENSMUSG00000031139 ) is located on Mouse chromosome X. 28 exons are identified, with the ATG start codon in exon 1 and the TAG stop codon in exon 28 (Transcript: ENSMUST00000063507). Exon 8 will be selected as conditional knockout region (cKO region). Deletion of this region should result in the loss of function of the Mouse Mcf2 gene. To engineer the targeting vector, homologous arms and cKO region will be generated by PCR using BAC clone RP23-87N13 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: Homozygous and hemizygous null mice are viable, fertile and behaviorally normal, exhibit normal gonad and brain development and neuronal migration, but show a significant reduction of basal dendritic length in distinct subpopulations of cortical pyramidal neurons. Exon 8 starts from about 24.18% of the coding region. The knockout of Exon 8 will result in frameshift of the gene. The size of intron 7 for 5'-loxP site insertion: 3430 bp, and the size of intron 8 for 3'-loxP site insertion: 632 bp. The size of effective cKO region: ~634 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 gRNA region 5' gRNA region 3' 1 8 9 28 Targeting vector Targeted allele Constitutive KO allele (After Cre recombination) Legends Exon of mouse Mcf2 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. 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(7134bp) | A(25.23% 1800) | C(21.35% 1523) | T(34.65% 2472) | G(18.77% 1339) 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% chrX - 60130428 60133427 3000 browser details YourSeq 532 1370 1918 3000 98.6% chr8 + 65730265 65730976 712 browser details YourSeq 525 1060 1806 3000 96.2% chr6 + 41274884 41275792 909 browser details YourSeq 521 1356 1918 3000 96.7% chr19 + 20373831 20374395 565 browser details YourSeq 517 1007 1806 3000 94.5% chr5 - 129773783 129774538 756 browser details YourSeq 500 1053 1806 3000 94.4% chr6 - 62371571 62372272 702 browser details YourSeq 492 1095 1806 3000 95.8% chrX + 81170286 81171211 926 browser details YourSeq 488 1059 1806 3000 96.4% chr9 - 96373011 96373931 921 browser details YourSeq 478 1006 1806 3000 93.6% chr7 - 123058926 123059516 591 browser details YourSeq 469 1050 1806 3000 97.0% chr10 - 23789389 23790311 923 browser details YourSeq 467 1028 1833 3000 97.4% chr1 + 184134087 184134984 898 browser details YourSeq 446 1368 1918 3000 94.9% chr19 - 34446994 34447468 475 browser details YourSeq 436 1358 1806 3000 98.7% chr10 + 93942815 93943271 457 browser details YourSeq 435 1356 1806 3000 98.0% chr18 - 50795448 50795897 450 browser details YourSeq 435 1356 1806 3000 97.8% chr1 - 74228995 74229444 450 browser details YourSeq 434 1356 1806 3000 97.6% chr15 - 21158686 21159126 441 browser details YourSeq 433 1364 1806 3000 98.9% chr10 - 73620100 73620542 443 browser details YourSeq 432 1329 1791 3000 97.2% chr3 - 130279348 130279812 465 browser details YourSeq 431 1349 1806 3000 97.4% chr9 + 85215928 85216403 476 browser details YourSeq 430 1358 1806 3000 98.0% chr12 - 51152501 51152969 469 Note: The 3000 bp section upstream of Exon 8 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% chrX - 60126794 60129793 3000 browser details YourSeq 157 1752 1954 3000 93.9% chrX + 113176717 113177154 438 browser details YourSeq 153 1720 1952 3000 85.6% chr14 - 54879375 54879620 246 browser details YourSeq 149 1783 1971 3000 94.7% chr5 - 92180715 92181123 409 browser details YourSeq 147 1782 1966 3000 95.1% chr15 - 37221303 37221491 189 browser details YourSeq 146 1782 1968 3000 93.5% chr10 - 93629340 93629531 192 browser details YourSeq 144 1762 1976 3000 91.8% chr5 + 139158204 139158728 525 browser details YourSeq 143 1782 1952 3000 94.0% chr12 + 102904191 102904378 188 browser details YourSeq 142 1781 1949 3000 95.0% chr9 + 26802935 26803117 183 browser details YourSeq 140 1782 1967 3000 94.3% chr3 + 127457841 127458044 204 browser details YourSeq 140 1783 1954 3000 92.7% chr17 + 74525844 74526029 186 browser details YourSeq 140 1706 1952 3000 91.7% chr17 + 47482817 47483371 555 browser details YourSeq 139 1782 1954 3000 93.8% chr5 - 18312596 18312786 191 browser details YourSeq 139 1783 1951 3000 92.6% chr14 + 34295579 34295781 203 browser details YourSeq 138 1780 1951 3000 93.1% chrX - 20665925 20666117 193 browser details YourSeq 138 1772 1952 3000 91.6% chr9 - 119115538 119115743 206 browser details YourSeq 138 1781 1952 3000 93.2% chr5 + 23696385 23696566 182 browser details YourSeq 137 1780 1952 3000 94.9% chr4 - 129837580 129837763 184 browser details YourSeq 137 1781 1952 3000 93.2% chr3 + 123502055 123502240 186 browser details YourSeq 136 1783 1952 3000 92.0% chr8 + 106627085 106627288 204 Note: The 3000 bp section downstream of Exon 8 is BLAT searched against the genome. No significant similarity is found. Page 4 of 7 https://www.alphaknockout.com Gene and protein information: Mcf2 mcf.2 transforming sequence [ Mus musculus (house mouse) ] Gene ID: 109904, updated on 12-Aug-2019 Gene summary Official Symbol Mcf2 provided by MGI Official Full Name mcf.2 transforming sequence provided by MGI Primary source MGI:MGI:96932 See related Ensembl:ENSMUSG00000031139 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 Dbl; Mcf-2; B230117G22Rik Expression Biased expression in cerebellum adult (RPKM 1.2), cortex adult (RPKM 0.7) and 9 other tissues See more Orthologs human all Genomic context Location: X A6; X 33.5 cM See Mcf2 in Genome Data Viewer Exon count: 30 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) X NC_000086.7 (60055956..60179184, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) X NC_000086.6 (57309133..57400820, complement) Chromosome X - NC_000086.7 Page 5 of 7 https://www.alphaknockout.com Transcript information: This gene has 4 transcripts Gene: Mcf2 ENSMUSG00000031139 Description mcf.2 transforming sequence [Source:MGI Symbol;Acc:MGI:96932] Gene Synonyms B230117G22Rik, Dbl Location Chromosome X: 60,055,956-60,179,089 reverse strand. GRCm38:CM001013.2 About this gene This gene has 4 transcripts (splice variants), 246 orthologues, 20 paralogues, is a member of 1 Ensembl protein family and is associated with 1 phenotype. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Mcf2-203 ENSMUST00000101531.9 3671 928aa ENSMUSP00000099070.3 Protein coding CCDS30160 Q8C067 TSL:1 GENCODE basic APPRIS P3 Mcf2-201 ENSMUST00000033478.4 3659 944aa ENSMUSP00000033478.4 Protein coding CCDS81129 A2AET8 TSL:1 GENCODE basic APPRIS ALT2 Mcf2-204 ENSMUST00000228150.1 3336 1111aa ENSMUSP00000154494.1 Protein coding CCDS72384 A0A2I3BR92 GENCODE basic APPRIS ALT2 Mcf2-202 ENSMUST00000063507.10 4227 1031aa ENSMUSP00000067075.4 Protein coding - Q8BLE2 TSL:1 GENCODE basic APPRIS ALT2 143.13 kb Forward strand 60.06Mb 60.08Mb 60.10Mb 60.12Mb 60.14Mb 60.16Mb 60.18Mb Genes Gm14891-201 >processed pseudogene (Comprehensive set... Contigs AL671984.12 > AL670778.15 > Genes < Gm5637-201processed pseudogene (Comprehensive set... < Mcf2-202protein coding < Mcf2-203protein coding < Mcf2-201protein coding < Mcf2-204protein coding Regulatory Build 60.06Mb 60.08Mb 60.10Mb 60.12Mb 60.14Mb 60.16Mb 60.18Mb Reverse strand 143.13 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding Ensembl protein coding merged Ensembl/Havana Non-Protein Coding pseudogene Page 6 of 7 https://www.alphaknockout.com Transcript: ENSMUST00000063507 < Mcf2-202protein coding Reverse strand 123.13 kb ENSMUSP00000067... Low complexity (Seg) Superfamily SSF46966 Dbl homology (DH) domain superfamily SSF50729 SMART CRAL-TRIO lipid binding domain Dbl homology (DH) domain Pleckstrin homology domain Pfam CRAL-TRIO lipid binding domain Dbl homology (DH) domain Pleckstrin homology domain PROSITE profiles CRAL-TRIO lipid binding domain Pleckstrin homology domain Dbl homology (DH) domain PROSITE patterns Guanine-nucleotide dissociation stimulator, CDC24, conserved site PANTHER PTHR22826:SF146 PTHR22826 Gene3D Dbl homology (DH) domain superfamily PH-like domain superfamily CDD CRAL-TRIO lipid binding domain Dbl homology (DH) domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend missense variant synonymous variant Scale bar 0 100 200 300 400 500 600 700 800 900 1031 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 7 of 7.
Load more

Recommended publications
  • Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes
    Genome-Wide Analysis of DNA Methylation Differences in Muscle and Fat from Monozygotic Twins Discordant for Type 2 Diabetes Rasmus Ribel-Madsen1,2*, Mario F. Fraga3, Stine Jacobsen1, Jette Bork-Jensen1, Ester Lara4, Vincenzo Calvanese4, Agustin F. Fernandez5, Martin Friedrichsen1, Birgitte F. Vind6, Kurt Højlund6, Henning Beck-Nielsen6, Manel Esteller7, Allan Vaag1,8, Pernille Poulsen9 1 Steno Diabetes Center, Gentofte, Denmark, 2 Section of Metabolic Genetics, The Novo Nordisk Foundation Center for Basic Metabolic Research, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark, 3 Department of Immunology and Oncology, National Centre for Biotechnology, CNB-CSIC, Madrid, Spain, 4 Department of Cancer Epigenetics, Spanish National Cancer Research Centre, Madrid, Spain, 5 Cancer Epigenetics Laboratory, Instituto Universitario de Oncologia del Principado de Asturias, HUCA, Universidad de Oviedo, Oviedo, Spain, 6 Department of Endocrinology, Odense University Hospital, Odense, Denmark, 7 Cancer Epigenetics and Biology Program (PEBC), Bellvitge Biomedical Research Institute (IDIBELL), L’Hospitalet de Llobregat, Barcelona, Spain, 8 Department of Endocrinology, Rigshospitalet, Copenhagen, Denmark, 9 Novo Nordisk A/S, Søborg, Denmark Abstract Background: Monozygotic twins discordant for type 2 diabetes constitute an ideal model to study environmental contributions to type 2 diabetic traits. We aimed to examine whether global DNA methylation differences exist in major glucose metabolic tissues from these twins. Methodology/Principal Findings: Skeletal muscle (n = 11 pairs) and subcutaneous adipose tissue (n = 5 pairs) biopsies were collected from 53–80 year-old monozygotic twin pairs discordant for type 2 diabetes. DNA methylation was measured by microarrays at 26,850 cytosine-guanine dinucleotide (CpG) sites in the promoters of 14,279 genes.
    [Show full text]
  • AN INVESTIGATION of the ROLE of PAK6 in TUMORIGENESIS By
    AN INVESTIGATION OF THE ROLE OF PAK6 IN TUMORIGENESIS by JoAnn Roberts A Thesis Submitted to the Faculty of The Charles E. Schmidt College of Medicine In Partial Fulfillment of the Requirements for the Degree of Master of Science Florida Atlantic University Boca Raton, Florida August 2012 ACKNOWLEDGMENTS This material is based upon work supported by the National Science Foundation under Grant No. DGE: 0638662. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. I would like to thank and acknowledge my thesis advisor, Dr. Michael Lu, for his support and guidance throughout the writing of this thesis and design of experiments in this manuscript. I would also like to thank my colleagues for assistance in various trouble-shooting circumstances. Last, but certainly not least, I would like to thank my family and friends for their support in the pursuit of my graduate studies. iii ABSTRACT Author: JoAnn Roberts Title: An Investigation of the Role of PAK6 in Tumorigenesis Institution: Florida Atlantic University Thesis Advisor: Dr. Michael Lu Degree: Master of Science Year: 2012 The function and role of PAK6, a serine/threonine kinase, in cancer progression has not yet been clearly identified. Several studies reveal that PAK6 may participate in key changes contributing to cancer progression such as cell survival, cell motility, and invasiveness. Based on the membrane localization of PAK6 in prostate and breast cancer cells, we speculated that PAK6 plays a role in cancer progression cells by localizing on the membrane and modifying proteins linked to motility and proliferation.
    [Show full text]
  • Src-Dependent DBL Family Members Drive Resistance to Vemurafenib in Human Melanoma Charlotte R
    Published OnlineFirst August 15, 2019; DOI: 10.1158/0008-5472.CAN-19-0244 Cancer Translational Science Research Src-Dependent DBL Family Members Drive Resistance to Vemurafenib in Human Melanoma Charlotte R. Feddersen1, Jacob L. Schillo1, Afshin Varzavand2, Hayley R. Vaughn1, Lexy S. Wadsworth1, Andrew P. Voigt1, Eliot Y. Zhu1, Brooke M. Jennings2, Sarah A. Mullen2, Jeremy Bobera2, Jesse D. Riordan1, Christopher S. Stipp2,3, and Adam J. Dupuy1,3 Abstract The use of selective BRAF inhibitors (BRAFi) has pro- signaling axis capable of driving resistance to both current duced remarkable outcomes for patients with advanced and next-generation BRAFis. However, we show that the cutaneous melanoma harboring a BRAFV600E mutation. SRC inhibitor, saracatinib, canblocktheDBL-drivenresis- Unfortunately, the majority of patients eventually develop tance. Our work highlights the utility of our straightforward drug-resistant disease. We employed a genetic screening genetic screening method in identifying new drug combina- approach to identify gain-of-function mechanisms of BRAFi tions to combat acquired BRAFi resistance. resistance in two independent melanoma cell lines. Our screens identified both known and unappreciated drivers of Significance: A simple, rapid, and flexible genetic screen- BRAFi resistance, including multiple members of the DBL ing approach identifies genes that drive resistance to MAPK family. Mechanistic studies identified a DBL/RAC1/PAK inhibitors when overexpressed in human melanoma cells. Introduction characterized (7). Thus, unexplained cases of resistance to MAPK inhibition (MAPKi) in human melanoma represent an important Melanoma is the deadliest form of skin cancer, with around unmet clinical need. 90,000 diagnoses of invasive disease and approximately 10,000 Mechanisms of vemurafenib resistance have been studied in deaths per year (1).
    [Show full text]
  • Yeast Artificial Chromosomes Spanning 8 Megabases and 10-15 Centimorgans of Human Cytogenetic Band Xq26 (Cloning/DNA/Genetic Map/Physical Map/Genome) RANDALL D
    Proc. Natl. Acad. Sci. USA Vol. 89, pp. 177-181, January 1992 Genetics Yeast artificial chromosomes spanning 8 megabases and 10-15 centimorgans of human cytogenetic band Xq26 (cloning/DNA/genetic map/physical map/genome) RANDALL D. LITrLE*, GIUSEPPE PILIA*, SANDRA JOHNSON*, MICHELE D'URSOt, AND DAVID SCHLESSINGER** *Department of Molecular Microbiology and Center for Genetics in Medicine, Washington University School of Medicine, St. Louis, MO 63110; and International Institute of Genetics and Biophysics, Naples, Italy 80125 Communicated by Norton D. Zinder, October 8, 1991 (receivedfor review August 10, 1991) ABSTRACT A successful test is reported to generate long- contig were obtained from a total human library (6), including range contiguous coverage of DNA from a human cytogenetic two clones, telomeric to probe a329R, which cover the only band in overlapping yeast artificial chromosomes (YACs). Seed segment not found in the Xq24-q28 collection. YACs are YACs in band Xq26 were recovered from a targeted library of named in accord with standard recommendations, with the clones from Xq24-q28 with 14 probes, including probes for the prefix y for YAC, W for Washington University in St. Louis, hypoxanthine guanine phosphoribosyltransferase- and coagu- XD for the laboratory of origin, and an accession number lation factor TX-encoding genes and nine probes used in linkage (XY837 in ref. 13, for example, is yWXD837 here). mapping. Neighboring YACs were then Identified by 25 "walk- Hybridization Probes Used to Organize the YAC Contig. All ing" steps with end-clones, and the content of 71 probes in probes were oligolabeled and hybridized to DNA in a matrix cognate YACs was verified by further hybridization analyses.
    [Show full text]
  • Targeting PH Domain Proteins for Cancer Therapy
    The Texas Medical Center Library DigitalCommons@TMC The University of Texas MD Anderson Cancer Center UTHealth Graduate School of The University of Texas MD Anderson Cancer Biomedical Sciences Dissertations and Theses Center UTHealth Graduate School of (Open Access) Biomedical Sciences 12-2018 Targeting PH domain proteins for cancer therapy Zhi Tan Follow this and additional works at: https://digitalcommons.library.tmc.edu/utgsbs_dissertations Part of the Bioinformatics Commons, Medicinal Chemistry and Pharmaceutics Commons, Neoplasms Commons, and the Pharmacology Commons Recommended Citation Tan, Zhi, "Targeting PH domain proteins for cancer therapy" (2018). The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access). 910. https://digitalcommons.library.tmc.edu/utgsbs_dissertations/910 This Dissertation (PhD) is brought to you for free and open access by the The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences at DigitalCommons@TMC. It has been accepted for inclusion in The University of Texas MD Anderson Cancer Center UTHealth Graduate School of Biomedical Sciences Dissertations and Theses (Open Access) by an authorized administrator of DigitalCommons@TMC. For more information, please contact [email protected]. TARGETING PH DOMAIN PROTEINS FOR CANCER THERAPY by Zhi Tan Approval page APPROVED: _____________________________________________ Advisory Professor, Shuxing Zhang, Ph.D. _____________________________________________
    [Show full text]
  • Genomic Studies of Motif Enrichment and Conservation in the Regulation
    Genomic Studies of Motif Enrichment and Conservation in the Regulation of Gene Expression in the Brain by David Alan Harmin B.A., Music and Physics, Wesleyan University (1976) JUN 2 0 2008 M.S., Physics, University of Chicago (1978) LIBRARIES Ph.D., Physics, University of Chicago (1981) Submitted to the Harvard-MIT Division of Health Sciences and Technology in partial fulfillment of the requirements for the degree of ARCHIV Master of Science in Biomedical Informatics at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY June 2008 @ Massachusetts Institute of Technology 2008. All rights reserved. Signature of Author ... ....... ......... ..... ......... Harvard-MIT ivision/of ea;h Sciences and Technology May 9, 2008 C ertified by ....... .t... 0. .U. .. ...............I s a "S. Kohane, M.D., Ph.D. Lawrence J. Henderson Associate Professor of Pediatrics and Health Sciences and Technology, HMS, CHMC Thesis Supervisor Accepted by. ............... V a/1Wa\tha L. Gray, Ph.D. Edwin Hood Taplin Professor of Medical and Ele trical Engineering, Director, Harvard-MIT Division of Health Sciences and Technology Genomic Studies of Motif Enrichment and Conservation in the Regulation of Gene Expression in the Brain by David Alan Harmin Submitted to the Harvard-MIT Division of Health Sciences and Technology on May 9, 2008, in partial fulfillment of the requirements for the degree of Master of Science in Biomedical Informatics Abstract Several bioinformatic tools will be brought to bear in this thesis to identify specific genomic loci that serve as regulatory gateways of gene expression in brain. These "motifs" are short nucleotide patterns that occur in promoters and 5' or 3' untrans- lated regions of genes.
    [Show full text]
  • A Novel Resveratrol Analog: Its Cell Cycle Inhibitory, Pro-Apoptotic and Anti-Inflammatory Activities on Human Tumor Cells
    A NOVEL RESVERATROL ANALOG : ITS CELL CYCLE INHIBITORY, PRO-APOPTOTIC AND ANTI-INFLAMMATORY ACTIVITIES ON HUMAN TUMOR CELLS A dissertation submitted to Kent State University in partial fulfillment of the requirements for the degree of Doctor of Philosophy by Boren Lin May 2006 Dissertation written by Boren Lin B.S., Tunghai University, 1996 M.S., Kent State University, 2003 Ph. D., Kent State University, 2006 Approved by Dr. Chun-che Tsai , Chair, Doctoral Dissertation Committee Dr. Bryan R. G. Williams , Co-chair, Doctoral Dissertation Committee Dr. Johnnie W. Baker , Members, Doctoral Dissertation Committee Dr. James L. Blank , Dr. Bansidhar Datta , Dr. Gail C. Fraizer , Accepted by Dr. Robert V. Dorman , Director, School of Biomedical Sciences Dr. John R. Stalvey , Dean, College of Arts and Sciences ii TABLE OF CONTENTS LIST OF FIGURES……………………………………………………………….………v LIST OF TABLES……………………………………………………………………….vii ACKNOWLEDGEMENTS….………………………………………………………….viii I INTRODUCTION….………………………………………………….1 Background and Significance……………………………………………………..1 Specific Aims………………………………………………………………………12 II MATERIALS AND METHODS.…………………………………………….16 Cell Culture and Compounds…….……………….…………………………….….16 MTT Cell Viability Assay………………………………………………………….16 Trypan Blue Exclusive Assay……………………………………………………...18 Flow Cytometry for Cell Cycle Analysis……………..……………....……………19 DNA Fragmentation Assay……………………………………………...…………23 Caspase-3 Activity Assay………………………………...……….….…….………24 Annexin V-FITC Staining Assay…………………………………..…...….………28 NF-kappa B p65 Activity Assay……………………………………..………….…29
    [Show full text]
  • Supplementary Materials
    Supplementary materials Supplementary Table S1: MGNC compound library Ingredien Molecule Caco- Mol ID MW AlogP OB (%) BBB DL FASA- HL t Name Name 2 shengdi MOL012254 campesterol 400.8 7.63 37.58 1.34 0.98 0.7 0.21 20.2 shengdi MOL000519 coniferin 314.4 3.16 31.11 0.42 -0.2 0.3 0.27 74.6 beta- shengdi MOL000359 414.8 8.08 36.91 1.32 0.99 0.8 0.23 20.2 sitosterol pachymic shengdi MOL000289 528.9 6.54 33.63 0.1 -0.6 0.8 0 9.27 acid Poricoic acid shengdi MOL000291 484.7 5.64 30.52 -0.08 -0.9 0.8 0 8.67 B Chrysanthem shengdi MOL004492 585 8.24 38.72 0.51 -1 0.6 0.3 17.5 axanthin 20- shengdi MOL011455 Hexadecano 418.6 1.91 32.7 -0.24 -0.4 0.7 0.29 104 ylingenol huanglian MOL001454 berberine 336.4 3.45 36.86 1.24 0.57 0.8 0.19 6.57 huanglian MOL013352 Obacunone 454.6 2.68 43.29 0.01 -0.4 0.8 0.31 -13 huanglian MOL002894 berberrubine 322.4 3.2 35.74 1.07 0.17 0.7 0.24 6.46 huanglian MOL002897 epiberberine 336.4 3.45 43.09 1.17 0.4 0.8 0.19 6.1 huanglian MOL002903 (R)-Canadine 339.4 3.4 55.37 1.04 0.57 0.8 0.2 6.41 huanglian MOL002904 Berlambine 351.4 2.49 36.68 0.97 0.17 0.8 0.28 7.33 Corchorosid huanglian MOL002907 404.6 1.34 105 -0.91 -1.3 0.8 0.29 6.68 e A_qt Magnogrand huanglian MOL000622 266.4 1.18 63.71 0.02 -0.2 0.2 0.3 3.17 iolide huanglian MOL000762 Palmidin A 510.5 4.52 35.36 -0.38 -1.5 0.7 0.39 33.2 huanglian MOL000785 palmatine 352.4 3.65 64.6 1.33 0.37 0.7 0.13 2.25 huanglian MOL000098 quercetin 302.3 1.5 46.43 0.05 -0.8 0.3 0.38 14.4 huanglian MOL001458 coptisine 320.3 3.25 30.67 1.21 0.32 0.9 0.26 9.33 huanglian MOL002668 Worenine
    [Show full text]
  • Tumorigenicity Assay. New Human Transforming Genes Detected by A
    New human transforming genes detected by a tumorigenicity assay. O Fasano, D Birnbaum, L Edlund, J Fogh and M Wigler Mol. Cell. Biol. 1984, 4(9):1695. DOI: 10.1128/MCB.4.9.1695. Downloaded from Updated information and services can be found at: http://mcb.asm.org/content/4/9/1695 These include: CONTENT ALERTS Receive: RSS Feeds, eTOCs, free email alerts (when new articles cite this article), more» http://mcb.asm.org/ on April 13, 2012 by MAIN LIBRARY Information about commercial reprint orders: http://mcb.asm.org/site/misc/reprints.xhtml To subscribe to to another ASM Journal go to: http://journals.asm.org/site/subscriptions/ MOLECULAR AND CELLULAR BIOLOGY, Sept. 1984, p. 1695-1705 Vol. 4, No. 9 0270-7306/84/091695-11$02.00/0 Copyright © 1984, American Society for Microbiology New Human Transforming Genes Detected by a Tumorigenicity Assay OTTAVIO FASANO,1 DANIEL BIRNBAUM,1 LOUISE EDLUND,' JORGEN FOGH,2 AND MICHAEL WIGLERl* Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724,1 and Sloan Kettering Institute, Rye, New York 105802 Received 20 April 1984/Accepted 1 June 1984 We have developed a sensitive bioassay for transforming genes based on the tumorigenicity of cotransfected Downloaded from NIH3T3 cells in nude mice. The assay differs substantially from the NIH3T3 focus assay. Using it, we have detected the transfer of three transforming genes from the DNA of MCF-7, a human mammary carcinoma cell line. One of these is N-ras, which is amplified in MCF-7 DNA. The other two, which we have called mcf2 and mcf3, do not appear to be related to known oncogenes.
    [Show full text]
  • Rho Guanine Nucleotide Exchange Factors: Regulators of Rho Gtpase Activity in Development and Disease
    Oncogene (2014) 33, 4021–4035 & 2014 Macmillan Publishers Limited All rights reserved 0950-9232/14 www.nature.com/onc REVIEW Rho guanine nucleotide exchange factors: regulators of Rho GTPase activity in development and disease DR Cook1, KL Rossman2,3 and CJ Der1,2,3 The aberrant activity of Ras homologous (Rho) family small GTPases (20 human members) has been implicated in cancer and other human diseases. However, in contrast to the direct mutational activation of Ras found in cancer and developmental disorders, Rho GTPases are activated most commonly in disease by indirect mechanisms. One prevalent mechanism involves aberrant Rho activation via the deregulated expression and/or activity of Rho family guanine nucleotide exchange factors (RhoGEFs). RhoGEFs promote formation of the active GTP-bound state of Rho GTPases. The largest family of RhoGEFs is comprised of the Dbl family RhoGEFs with 70 human members. The multitude of RhoGEFs that activate a single Rho GTPase reflects the very specific role of each RhoGEF in controlling distinct signaling mechanisms involved in Rho activation. In this review, we summarize the role of Dbl RhoGEFs in development and disease, with a focus on Ect2 (epithelial cell transforming squence 2), Tiam1 (T-cell lymphoma invasion and metastasis 1), Vav and P-Rex1/2 (PtdIns(3,4,5)P3 (phosphatidylinositol (3,4,5)-triphosphate)-dependent Rac exchanger). Oncogene (2014) 33, 4021–4035; doi:10.1038/onc.2013.362; published online 16 September 2013 Keywords: Rac1; RhoA; Cdc42; guanine nucleotide exchange factors; cancer;
    [Show full text]
  • MCF2 Is Linked to a Complex Perisylvian Syndrome and Affects Cortical Lamination Running Head
    bioRxiv preprint doi: https://doi.org/10.1101/733444; this version posted August 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Title: MCF2 is linked to a complex perisylvian syndrome and affects cortical lamination Running head: Perisylvian syndrome, cortical lamination and MCF2 Authors: Aude Molinard-Chenu, PhD1,2,3, Joël Fluss, MD6, Sacha Laurent, PhD4, Michel Guipponi, PhD4,5, Alexandre G Dayer, MD 1,2,3 Corresponding Author: Aude Molinard-Chenu Number of characters in the title: 80 (including spaces), 69 (excluding spaces) Number of characters in the running head: 50 (including spaces), 45 (excluding spaces) Number of words: 100 (Abstract), 1436 (Body of the manuscript) Number of figures: 2 (1 B/W figure and 1 color figure) 1 Department of Psychiatry, University of Geneva Medical School, Geneva 4 CH-1211, Switzerland. 2 Department of Basic Neurosciences, University of Geneva Medical School, Geneva 4 CH- 1211, Switzerland. 3 Institute of Genetics and Genomics in Geneva (IGe3), University of Geneva Medical Center (CMU), Geneva 4 CH – 1211, Switzerland 4 Service of Genetic Medicine, University Hospitals of Geneva, Geneva, Switzerland 5 Department of Genetic Medicine and Development, University of Geneva Medical School, Geneva, Switzerland. 6 Pediatric Neurology Unit, Pediatric Subspecialties Service, University Hospitals of Geneva, Geneva, Switzerland bioRxiv preprint doi: https://doi.org/10.1101/733444; this version posted August 13, 2019. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
    [Show full text]
  • CCPG1 (NM 001204451) Human Tagged ORF Clone Product Data
    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 RG234057 CCPG1 (NM_001204451) Human Tagged ORF Clone Product data: Product Type: Expression Plasmids Product Name: CCPG1 (NM_001204451) Human Tagged ORF Clone Tag: TurboGFP Symbol: CCPG1 Synonyms: CPR8 Vector: pCMV6-AC-GFP (PS100010) E. coli Selection: Ampicillin (100 ug/mL) Cell Selection: Neomycin ORF Nucleotide >RG234057 representing NM_001204451 Sequence: Red=Cloning site Blue=ORF Green=Tags(s) TTTTGTAATACGACTCACTATAGGGCGGCCGGGAATTCGTCGACTGGATCCGGTACCGAGGAGATCTGCC GCCGCGATCGCC ATGTCTGAAAATTCCAGTGACAGTGATTCATCTTGTGGTTGGACTGTCATCAGTCATGAGGGGTCAGATA TAGAAATGTTGAATTCTGTGACCCCCACTGACAGCTGTGAGCCCGCCCCAGAATGTTCATCTTTAGAGCA AGAGGAGCTTCAAGCATTGCAGATAGAGCAAGGAGAAAGCAGCCAAAATGGCACAGTGCTTATGGAAGAA ACTGCTTATCCAGCTTTGGAGGAAACCAGCTCAACAATTGAGGCAGAGGAACAAAAGATACCCGAAGACA GTATCTATATTGGAACTGCCAGTGATGATTCTGATATTGTTACCCTTGAGCCACCTAAGTTAGAAGAAAT TGGAAATCAAGAAGTTGTCATTGTTGAAGAAGCACAGAGTTCAGAAGACTTTAACATGGGCTCTTCCTCT AGCAGCCAGTATACTTTCTGTCAGCCAGAAACTGTATTTTCATCTCAGCCTAGTGACGATGAATCAAGTA GTGATGAAACCAGTAATCAGCCCAGTCCTGCCTTTAGACGACGCCGTGCTAGGAAGAAGACCGTTTCTGC TTCAGAATCTGAAGACCGGCTAGTTGCTGAACAAGAAACTGAACCTTCTAAGGAGTTGAGTAAACGTCAG TTCAGTAGTGGTCTCAATAAGTGTGTTATACTTGCTTTGGTGATTGCAATCAGCATGGGATTTGGCCATT TCTATGGCACAATTCAGATTCAGAAGCGTCAACAGTTAGTCAGAAAGATACATGAAGATGAATTGAATGA TATGAAGGATTATCTTTCCCAGTGTCAACAGGAACAAGAATCTTTTATAGATTATAAGTCATTGAAAGAA AATCTTGCAAGGTGTTGGACACTTACTGAAGCAGAGAAGATGTCCTTTGAAACTCAGAAAACGAACCTTG
    [Show full text]