Mouse Fgl1 Knockout Project (CRISPR/Cas9)

Total Page:16

File Type:pdf, Size:1020Kb

Mouse Fgl1 Knockout Project (CRISPR/Cas9) https://www.alphaknockout.com Mouse Fgl1 Knockout Project (CRISPR/Cas9) Objective: To create a Fgl1 knockout Mouse model (C57BL/6N) by CRISPR/Cas-mediated genome engineering. Strategy summary: The Fgl1 gene (NCBI Reference Sequence: NM_145594 ; Ensembl: ENSMUSG00000031594 ) is located on Mouse chromosome 8. 8 exons are identified, with the ATG start codon in exon 2 and the TAG stop codon in exon 8 (Transcript: ENSMUST00000034003). Exon 3~4 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 one null allele exhibit increased body weight, white fat and gluconeogenesis, decreased circulating cholesterol, free fatty acid level and respiratory quotient, hyperglycemia, and impaired glucose tolerance. Mice homozygous for a second null allele display normal appearance with age-related onset of dermatitis. Exon 3 starts from about 6.79% of the coding region. Exon 3~4 covers 36.84% of the coding region. The size of effective KO region: ~9439 bp. The KO region does not have any other known gene. Page 1 of 8 https://www.alphaknockout.com Overview of the Targeting Strategy Wildtype allele 5' gRNA region gRNA region 3' 1 3 4 8 Legends Exon of mouse Fgl1 Knockout region Page 2 of 8 https://www.alphaknockout.com Overview of the Dot Plot (up) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 543 bp section upstream of Exon 3 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 Dot Plot (down) Window size: 15 bp Forward Reverse Complement Sequence 12 Note: The 695 bp section downstream of Exon 4 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 8 https://www.alphaknockout.com Overview of the GC Content Distribution (up) Window size: 300 bp Sequence 12 Summary: Full Length(543bp) | A(33.7% 183) | C(17.86% 97) | T(29.47% 160) | G(18.97% 103) Note: The 543 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(695bp) | A(22.3% 155) | C(17.41% 121) | T(38.13% 265) | G(22.16% 154) Note: The 695 bp section downstream of Exon 4 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 8 https://www.alphaknockout.com BLAT Search Results (up) QUERY SCORE START END QSIZE IDENTITY CHROM STRAND START END SPAN ----------------------------------------------------------------------------------------------- browser details YourSeq 543 1 543 543 100.0% chr8 - 41209884 41210426 543 browser details YourSeq 36 39 76 543 100.0% chr11 + 55458536 55770756 312221 browser details YourSeq 28 201 229 543 100.0% chr15 - 38662546 38662575 30 browser details YourSeq 25 155 180 543 100.0% chr19 - 36841069 36841097 29 browser details YourSeq 25 22 47 543 100.0% chr17 + 28478295 28478323 29 browser details YourSeq 24 136 162 543 96.3% chr1 - 134696895 134696922 28 browser details YourSeq 24 41 67 543 96.3% chr2 + 8425896 8425925 30 browser details YourSeq 23 41 64 543 100.0% chr2 - 84108928 84108956 29 browser details YourSeq 23 169 195 543 92.6% chr2 + 32275926 32275952 27 browser details YourSeq 21 181 203 543 95.7% chr2 - 29096900 29096922 23 browser details YourSeq 21 174 194 543 100.0% chr12 - 5458196 5458216 21 browser details YourSeq 20 41 60 543 100.0% chr11 + 64184901 64184920 20 browser details YourSeq 20 53 72 543 100.0% chr10 + 20417547 20417566 20 Note: The 543 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 695 1 695 695 100.0% chr8 - 41199750 41200444 695 browser details YourSeq 235 86 500 695 87.5% chr4 + 129411747 129412289 543 browser details YourSeq 227 87 500 695 86.5% chrX + 101498396 101499015 620 browser details YourSeq 216 78 500 695 85.7% chr7 - 13059392 13060001 610 browser details YourSeq 215 125 500 695 82.9% chr17 + 28526319 28526647 329 browser details YourSeq 203 67 500 695 81.9% chr5 + 65397076 65397472 397 browser details YourSeq 196 132 500 695 86.7% chr5 + 34391098 34671465 280368 browser details YourSeq 194 138 487 695 87.4% chr3 - 58579777 58580410 634 browser details YourSeq 187 78 500 695 82.2% chr9 + 66602571 66602955 385 browser details YourSeq 179 188 500 695 86.8% chr10 + 40407200 40407483 284 browser details YourSeq 171 115 500 695 84.9% chr6 + 148987454 148987813 360 browser details YourSeq 166 74 500 695 90.7% chr4 + 45340042 45340539 498 browser details YourSeq 159 188 500 695 90.0% chr17 - 24458495 24807709 349215 browser details YourSeq 154 110 514 695 89.3% chr17 - 83612055 83612643 589 browser details YourSeq 152 336 514 695 92.8% chr7 + 80289410 80289591 182 browser details YourSeq 150 202 500 695 91.8% chr17 - 85054234 85054574 341 browser details YourSeq 148 46 500 695 88.3% chr7 + 127639949 127640482 534 browser details YourSeq 146 336 531 695 89.3% chr12 + 59167277 59167473 197 browser details YourSeq 141 235 492 695 92.3% chr3 - 58365711 58366061 351 browser details YourSeq 141 339 510 695 91.3% chr11 - 26541347 26541709 363 Note: The 695 bp section downstream of Exon 4 is BLAT searched against the genome. No significant similarity is found. Page 5 of 8 https://www.alphaknockout.com Gene and protein information: Fgl1 fibrinogen-like protein 1 [ Mus musculus (house mouse) ] Gene ID: 234199, updated on 12-Aug-2019 Gene summary Official Symbol Fgl1 provided by MGI Official Full Name fibrinogen-like protein 1 provided by MGI Primary source MGI:MGI:102795 See related Ensembl:ENSMUSG00000031594 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 Mfire1 Expression Restricted expression toward liver E18 (RPKM 174.3) See more Orthologs human all Genomic context Location: 8; 8 A4 See Fgl1 in Genome Data Viewer Exon count: 9 Annotation release Status Assembly Chr Location 108 current GRCm38.p6 (GCF_000001635.26) 8 NC_000074.6 (41191434..41217978, complement) Build 37.2 previous assembly MGSCv37 (GCF_000001635.18) 8 NC_000074.5 (42276788..42300510, complement) Chromosome 8 - NC_000074.6 Page 6 of 8 https://www.alphaknockout.com Transcript information: This gene has 2 transcripts Gene: Fgl1 ENSMUSG00000031594 Description fibrinogen-like protein 1 [Source:MGI Symbol;Acc:MGI:102795] Location Chromosome 8: 41,191,434-41,215,156 reverse strand. GRCm38:CM001001.2 About this gene This gene has 2 transcripts (splice variants), 130 orthologues, 23 paralogues, is a member of 1 Ensembl protein family and is associated with 26 phenotypes. Transcripts Name Transcript ID bp Protein Translation ID Biotype CCDS UniProt Flags Fgl1-201 ENSMUST00000034003.4 1120 314aa ENSMUSP00000034003.4 Protein coding CCDS22260 A0A0R4J0E1 TSL:1 GENCODE basic APPRIS P1 Fgl1-202 ENSMUST00000134510.1 3907 No protein - Retained intron - - TSL:2 43.72 kb Forward strand 41.19Mb 41.20Mb 41.21Mb 41.22Mb Genes Gm16348-202 >lncRNA (Comprehensive set... Gm16348-201 >lncRNA Contigs < AC156554.13 Genes (Comprehensive set... < Fgl1-201protein coding < Fgl1-202retained intron Regulatory Build 41.19Mb 41.20Mb 41.21Mb 41.22Mb Reverse strand 43.72 kb Regulation Legend CTCF Open Chromatin Promoter Promoter Flank Gene Legend Protein Coding merged Ensembl/Havana Non-Protein Coding RNA gene processed transcript Page 7 of 8 https://www.alphaknockout.com Transcript: ENSMUST00000034003 < Fgl1-201protein coding Reverse strand 23.72 kb ENSMUSP00000034... Coiled-coils (Ncoils) Cleavage site (Sign... Superfamily Fibrinogen-like, C-terminal SMART Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain Pfam Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain PROSITE profiles Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain PROSITE patterns Fibrinogen, conserved site PANTHER PTHR19143 PTHR19143:SF263 Gene3D 3.90.215.20 CDD Fibrinogen, alpha/beta/gamma chain, C-terminal globular domain All sequence SNPs/i... Sequence variants (dbSNP and all other sources) Variant Legend inframe deletion missense variant synonymous variant Scale bar 0 40 80 120 160 200 240 314 We wish to acknowledge the following valuable scientific information resources: Ensembl, MGI, NCBI, UCSC. Page 8 of 8.
Recommended publications
  • LAG-3: from Molecular Functions to Clinical Applications
    Open access Review J Immunother Cancer: first published as 10.1136/jitc-2020-001014 on 13 September 2020. Downloaded from LAG-3: from molecular functions to clinical applications Takumi Maruhashi , Daisuke Sugiura , Il- mi Okazaki , Taku Okazaki To cite: Maruhashi T, Sugiura D, ABSTRACT (PD-1) and cytotoxic T lymphocyte antigen Okazaki I, et al. LAG-3: from To prevent the destruction of tissues owing to excessive 4 (CTLA-4) significantly improved the molecular functions to clinical and/or inappropriate immune responses, immune outcomes of patients with diverse cancer applications. Journal for cells are under strict check by various regulatory ImmunoTherapy of Cancer types, revolutionizing cancer treatment. The mechanisms at multiple points. Inhibitory coreceptors, 2020;8:e001014. doi:10.1136/ success of these therapies verified that inhib- including programmed cell death 1 (PD-1) and cytotoxic jitc-2020-001014 itory coreceptors serve as critical checkpoints T lymphocyte antigen 4 (CTLA-4), serve as critical checkpoints in restricting immune responses against for immune cells to not attack the tumor Accepted 29 July 2020 self- tissues and tumor cells. Immune checkpoint inhibitors cells as well as self-tissues. However, response that block PD-1 and CTLA-4 pathways significantly rates are typically lower and immune-related improved the outcomes of patients with diverse cancer adverse events (irAEs) are also observed in types and have revolutionized cancer treatment. However, patients administered with immune check- response rates to such therapies are rather limited, and point inhibitors. This is indicative of the immune-rela ted adverse events are also observed in a continued need to decipher the complex substantial patient population, leading to the urgent need biology of inhibitory coreceptors to increase for novel therapeutics with higher efficacy and lower response rates and prevent such unwanted toxicity.
    [Show full text]
  • Cytogenetic and Molecular Genetic Alterations in Hepatocellular Carci- Noma
    Acta Pharmacologica Sinica 2005 Jun; 26 (6): 659–665 Invited review Cytogenetic and molecular genetic alterations in hepatocellular carci- noma Sze-hang LAU, Xin-yuan GUAN1 Department of Clinical Oncology, Faculty of Medicine, The University of Hong Kong, Hong Kong, China Key words Abstract hepatocellular carcinoma; chromosome Specific chromosome aberrations are frequently detected during the development aberrations; oncogenes; tumor suppressor of hepatocellular carcinoma. Molecular cytogenetic approaches such as com- genes parative genomic hybridization and loss of heterozygosity analyses have pro- vided fruitful information on changes in HCC cases at the genomic level. Map- 1 Correspondence to Dr Xin-yuan GUAN. ping of chromosome gains and losses have frequently resulted in the identifica- Fax 852-2816-9126. E-mail [email protected] tion of oncogenes and tumor suppressors, respectively. In this review, we sum- marize some frequently detected chromosomal aberrations reported for hepatocel- Received 2005-02-17 lular carcinoma cases using comparative genomic hybridization and loss of het- Accepted 2005-03-25 erozygosity studies. Focus will be on gains of 1q, 8q, and 20q, and losses of 4q, doi: 10.1111/j.1745-7254.2005.00126.x 8p, 13q, 16q, and 17p. We then examine the candidate oncogenes and tumor suppressors located within these regions, and explore their possible functions in hepatocarcinogenesis. Finally, the impact of microarray-based screening platforms will be discussed. Introduction based on the observation of chromosome deletion del(13) (q14) in retinoblastoma[6] and the proto-oncogene myc was Hepatocellular carcinoma (HCC) is one of the most com- shown to be involved in the chromosome translocation t(8; mon human malignant neoplasms, with a particularly high 14) in human Burkett’s lymphoma[7].
    [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]
  • New Ligand for LAG-3 Vaccine Responses
    research highlights TRM CELLS In Cell, Chen and colleagues identify the dependent insulinotropic polypeptide Tissue adaptation fibrinogen family protein FGL1 as a major (GIP) signaling and the suppression Science https://doi.org/10.1126/science.aat6280 ligand for LAG-3. The FGL1–LAG-3 of myeloid cell inflammatory responses. (2018) interaction is conserved in human and Mice that lack expression of the GIP mouse, is specific to FGL1, involves the receptor specifically in myeloid cells Commensals and commensal-reactive fibrinogen-like domain of FGL1 and exhibit excessive weight gain, impaired the D1-D2 domain of LAG-3 and is glucose tolerance and dysregulation lymphocytes coexist at barrier tissues. In –/– Science, Belkaid and colleagues show that independent of MHC class II. Fgl1 mice of cold-induced adaptive thermogenesis develop spontaneous autoimmunity with when fed a high-fat diet. Lack of skin-resident commensal-specific T cells –/– –/– express a type 17 program associated with age. Similar to Lag3 mice, Fgl1 mice the GIP receptor leads to increased a poised type 2 program. Staphylococcus control the growth of inoculated tumors expression of the alarmin S100A8 epidermis–colonized mice develop long- better than wild-type mice do, in a manner by fat-resident myeloid cells. This + + lived, tissue-resident, S. epidermis–specific dependent on CD8 T cells and CD4 scenario leads to greater myelopoiesis, + T cells. Antibodies to LAG-3 are not neutrophilia and recruitment of CD8 memory T cells. S. epidermis–elicited –/– + + + + protective against tumors in the Fgl1 myeloid cells to fat depots than RORγ t CD4 TH17 cells and RORγ t CD8 Tc17 cells produce the type 2 cytokines IL-5 mice.
    [Show full text]
  • Development and Characterization of Immunogenic Genetically Engineered Mouse Models of Pancreatic Cancer
    Development and characterization of immunogenic genetically engineered mouse models of pancreatic cancer By Laurens J. Lambert MSc, Medical Biology Radboud University, 2014 Submitted to the Department of Biology in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy at the MASSACHUSETTS INSTITUTE OF TECHNOLOGY September 2020 © 2020 Massachusetts Institute of Technology. All rights reserved. Signature of Author………………………………………………………………………………. Laurens J. Lambert Department of Biology June 16, 2020 Certified by………..………………………………………………………………………………. Tyler Jacks David H. Koch Professor of Biology Investigator, Howard Hughes Medical Institute Thesis Supervisor Accepted by………………………………………………………………………………………. Stephen Bell Uncas and Helen Whitaker Professor of Biology Investigator, Howard Hughes Medical Institute Co-Director, Biology Graduate Committee 2 Development and characterization of immunogenic genetically engineered mouse models of pancreatic cancer By Laurens J. Lambert Submitted to the Department of Biology on June 16, 2020 in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Biology Abstract Insights into mechanisms of immune escape have fueled the clinical success of immunotherapy in many cancers. However, pancreatic cancer has remained largely refractory to checkpoint immunotherapy. To uncover mechanisms of immune escape, we have characterized two preclinical models of immunogenic pancreatic ductal adenocarcinoma (PDAC). In order to dissect the endogenous antigen-specific T cell response in PDAC, lentivirus encoding the Cre recombinase and a tumor specific antigen LSL-G12D/+; flox/flox (SIINFEKL, OVA257-264) was delivered to Kras Trp53 (KP) mice. We demonstrate that KP tumors show distinct antigenic outcomes: a subset of PDAC tumors undergoes clearance or editing by a robust antigen-specific CD8+ T cell response, while a fraction undergo immune escape.
    [Show full text]
  • Investigation of the Association Between FGL1 Expression and Prognosis in Gastric Cancer Patients Mahnaz Saremi1*, Leila Moezzi2
    http://pmjournal.ir Original Article Autumn 2020, Volume 5, Issue 19 (7-9) Investigation of the Association between FGL1 Expression and Prognosis in Gastric Cancer Patients Mahnaz Saremi1*, Leila Moezzi2 1 Reference Health Laboratory, Ministry of Health and Medical Education 2 Department of Cellular and Molecular, Faculty of Life Sciences, North Tehran Branch, Islamic Azad University, Faculty of Biological Sciences, Tehran, Iran 2 Personalized Medicine Research Center of AmitisGen, Tehran, Iran *Corresponding author: Mahnaz Saremi, Reference Health Laboratory, Ministry of Health and Medical DOI: 10.22034/pmj.2020.240044 Education. Email: :[email protected] Submitted: 2020/05/23 Abstract Accepted: 2020/07/19 Gastric cancer is the fourth most common cancer worldwide, and it ranks second leading Keywords: cause of cancer deaths. Several studies have shown that FGL2 contributes to the patho- Gastric cancer genesis of a number of infectious diseases. However, little is known about its biological FGL2 gene functions in cancer development and metastasis. In this study, the association between gene expression FGL1 expression and prognosis was investigated in GC patients. Gastric cancer and ad- qPCR jacent normal tissues (n=20) were obtained from patients diagnosed with gastric cancer aged between 30 and 50. Total RNA was extracted, reverse transcription and qPCR were ©2020.Personalized Medicine Journal performed, and Relative expression level was calculated using the 2-∆∆Cq method. It was found that FGL1 expression in gastric cancer tissues was obviously higher than adjacent tissues at mRNA levels (P<0.003). IntroductIon effector molecule of Treg cells and plays a critical Gastric cancer is the fourth most common role in regulating innate immunity and adaptive cancer worldwide, and it ranks as the second immunity [7].
    [Show full text]
  • The Role of Fibrinogen-Like Proteins in Cancer
    The Role of Fibrinogen-Like Proteins in Cancer Jing Yu1,2#, Jing Li 3#, Jing Shen1,2, Fukuan Du1,2, Xu Wu1,2, Mingxing Li1,2, Yu Chen1,2, Chi Hin Cho1,2, Xiaobing Li1*, Zhangang Xiao1,2*, Yueshui Zhao1,2* 1. Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China 2. South Sichuan Institute of Translational Medicine, Luzhou, Sichuan, China 3. Department of Oncology and Hematology, Hospital (T.C.M) Affiliated to Southwest Medical University, Luzhou, Sichuan, China. # These Authors Contributed Equally to This Work. *Addresses for Correspondent Authors: Yueshui Zhao, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, China; E-mail: [email protected] Zhangang Xiao, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, China; E-mail: [email protected] Xiaobing Li, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, China; E-mail: [email protected] 1 Abstract Fibrinogen-associated protein (FREP) family is a family of proteins with a fibrin domain at the carboxyl terminus. Recent investigations illustrated that two members of FREP family, fibrinogen-like protein-1 (FGL1) and fibrinogen-like protein-2 (FGL2), play crucial roles in cancer by regulating the proliferation, invasion, and migration of tumor cells, or regulating the functions of immune cells in tumor microenvironment. Meanwhile, they are potential targets for medical intervention of tumor development. In this review, we discussed the structure, and the roles of FGL1 and FGL2 in tumors, especially the roles in regulating immune cell functions.
    [Show full text]
  • The Role of Fibrinogen-Like Proteins in Cancer
    Int. J. Biol. Sci. 2021, Vol. 17 1079 Ivyspring International Publisher International Journal of Biological Sciences 2021; 17(4): 1079-1087. doi: 10.7150/ijbs.56748 Review The role of Fibrinogen-like proteins in Cancer Jing Yu1,2#, Jing Li3#, Jing Shen1,2, Fukuan Du1,2, Xu Wu1,2, Mingxing Li1,2, Yu Chen1,2, Chi Hin Cho1,2, Xiaobing Li1, Zhangang Xiao1,2 and Yueshui Zhao1,2,4 1. Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan, China. 2. South Sichuan Institute of Translational Medicine, Luzhou, Sichuan, China. 3. Department of Oncology and Hematology, Hospital (T.C.M) Affiliated to Southwest Medical University, Luzhou, Sichuan, China. 4. Department of Pharmacy, The Affiliated Hospital of Southwest Medical University, Luzhou, Sichuan, China. # These authors contributed equally to this work. Corresponding authors: Yueshui Zhao, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University; South Sichuan Institute of Translational Medicine; Department of Pharmacy, The Affiliated Hospital of Southwest Medical University; Luzhou, Sichuan 646000, China. E-mail: [email protected]; Zhangang Xiao, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University; South Sichuan Institute of Translational Medicine; Luzhou, Sichuan 646000, China. E-mail: [email protected]; Xiaobing Li, Laboratory of Molecular Pharmacology, Department of Pharmacology, School of Pharmacy, Southwest Medical University, Luzhou, Sichuan 646000, China. E-mail: [email protected]. © The author(s). This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
    [Show full text]
  • FGL1 CRISPR/Cas9 KO Plasmid (M): Sc-433343
    SANTA CRUZ BIOTECHNOLOGY, INC. FGL1 CRISPR/Cas9 KO Plasmid (m): sc-433343 BACKGROUND APPLICATIONS The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and FGL1 CRISPR/Cas9 KO Plasmid (m) is recommended for the disruption of CRISPR-associated protein (Cas9) system is an adaptive immune response gene expression in mouse cells. defense mechanism used by archea and bacteria for the degradation of foreign genetic material (4,6). This mechanism can be repurposed for other 20 nt non-coding RNA sequence: guides Cas9 functions, including genomic engineering for mammalian systems, such as to a specific target location in the genomic DNA gene knockout (KO) (1,2,3,5). CRISPR/Cas9 KO Plasmid products enable the U6 promoter: drives gRNA scaffold: helps Cas9 identification and cleavage of specific genes by utilizing guide RNA (gRNA) expression of gRNA bind to target DNA sequences derived from the Genome-scale CRISPR Knock-Out (GeCKO) v2 library developed in the Zhang Laboratory at the Broad Institute (3,5). Termination signal Green Fluorescent Protein: to visually REFERENCES verify transfection CRISPR/Cas9 Knockout Plasmid CBh (chicken β-Actin 1. Cong, L., et al. 2013. Multiplex genome engineering using CRISPR/Cas hybrid) promoter: drives systems. Science 339: 819-823. 2A peptide: expression of Cas9 allows production of both Cas9 and GFP from the 2. Mali, P., et al. 2013. RNA-guided human genome engineering via Cas9. same CBh promoter Science 339: 823-826. Nuclear localization signal 3. Ran, F.A., et al. 2013. Genome engineering using the CRISPR-Cas9 system. Nuclear localization signal SpCas9 ribonuclease Nat. Protoc. 8: 2281-2308.
    [Show full text]
  • A Cluster of Cooperating Tumor-Suppressor Gene Candidates in Chromosomal Deletions
    A cluster of cooperating tumor-suppressor gene candidates in chromosomal deletions Wen Xuea,1,2, Thomas Kitzinga,1,3, Stephanie Roesslerb, Johannes Zubera, Alexander Krasnitza, Nikolaus Schultzc, Kate Revilla, Susann Weissmuellerd,3, Amy R. Rappaporta, Janelle Simona,e,3, Jack Zhanga, Weijun Luoa, James Hicksa, Lars Zendera,4, Xin Wei Wangb, Scott Powersa, Michael Wiglera,5, and Scott W. Lowea,e,3,5 aCold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; bLaboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; cComputational Biology Center, Memorial Sloan-Kettering Cancer Center, New York, NY 10065; dWatson School of Biological Sciences, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724; and eHoward Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, NY 10065 Contributed by Michael Wigler, April 12, 2012 (sent for review January 13, 2012) The large chromosomal deletions frequently observed in cancer the extent of chromosome 8p deletions from cancer genome genomes are often thought to arise as a “two-hit” mechanism in the datasets derived from array-based comparative genomic hybrid- process of tumor-suppressor gene (TSG) inactivation. Using a murine ization (aCGH) performed at Cold Spring Harbor Laboratory and model system of hepatocellular carcinoma (HCC) and in vivo RNAi, the Cancer Genome Atlas (TCGA) project, totaling 1411 primary we test an alternative hypothesis, that such deletions can arise from tumor samples and cell lines of HCC and breast, colon, and lung selective pressure to attenuate the activity of multiple genes. By cancers (Fig. 1A and Materials and Methods). According to these targeting the mouse orthologs of genes frequently deleted on hu- data, approximately half of these tumors harbor heterozygous man 8p22 and adjacent regions, which are lost in approximately deletions of human chromosome 8p, often encompassing a large half of several other major epithelial cancers, we provide evidence portion of or even the entire chromosome arm (Fig.
    [Show full text]
  • Fibrinogen-Like Protein 1 Modulates Sorafenib Resistance in Human Hepatocellular Carcinoma Cells
    International Journal of Molecular Sciences Article Fibrinogen-Like Protein 1 Modulates Sorafenib Resistance in Human Hepatocellular Carcinoma Cells Yeonghoon Son 1,2,†, Na-Rae Shin 1,†, Sung-Ho Kim 3, Su-Cheol Park 4 and Hae-June Lee 1,* 1 Division of Basic Radiation Bioscience, Korea Institute of Radiological & Medical Sciences, Seoul 01812, Korea; [email protected] (Y.S.); [email protected] (N.-R.S.) 2 Primate Resources Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Jeonbuk 56216, Korea 3 College of Veterinary Medicine, Chonnam National University, Gwangju 61186, Korea; [email protected] 4 Department of Internal Medicine, Korea Cancer Center Hospital, Korea Institute of Radiological and Medical Sciences, Seoul 01812, Korea; [email protected] * Correspondence: [email protected]; Tel.: +82-2-970-1638; Fax: +82-2-970-1985 † These authors contributed equally. Abstract: Despite liver cancer being the second-leading cause of cancer-related death worldwide, few systemic drugs have been approved. Sorafenib, the first FDA-approved systemic drug for unre- sectable hepatocellular carcinoma (HCC), is limited by resistance. However, the precise mechanisms underlying this phenomenon are unknown. Since fibrinogen-like 1 (FGL1) is involved in HCC progression and upregulated after anticancer therapy, we investigated its role in regulating sorafenib resistance in HCC. FGL1 expression was assessed in six HCC cell lines (HepG2, Huh7, Hep3B, SNU387, SNU449, and SNU475) using western blotting. Correlations between FGL1 expression and sorafenib resistance were examined by cell viability, colony formation, and flow cytometry Citation: Son, Y.; Shin, N.-R.; Kim, assays. FGL1 was knocked-down to confirm its effects on sorafenib resistance.
    [Show full text]
  • Data Sheet FGL1:LAG3 TR-FRET Assay Kit Catalog #79739-1 Size: 96 Reactions
    6042 Cornerstone Court W, Ste B San Diego, CA 92121 Tel: 1.858.202.1401 Fax: 1.858.481.8694 Email: [email protected] Data sheet FGL1:LAG3 TR-FRET Assay Kit Catalog #79739-1 Size: 96 reactions BACKGROUND: Lymphocyte-activation gene 3 (LAG3, also CD223) is a cell surface receptor that negatively regulates activation and proliferation of T cells. Fibrinogen-like protein 1 (FGL1), a liver-secreted protein, is a functional LAG3 ligand. Blockade of the FGL1-LAG3 interaction is implicated in promoting antitumor immunity. DESCRIPTION: The FGL1:LAG3 TR-FRET Assay is designed to measure the inhibition of LAG3 binding to FGL1 in a homogeneous 96 reaction format. This TR-FRET-based assay requires no time-consuming washing steps, making it especially suitable for high throughput screening applications. The assay procedure is straightforward and simple; a sample containing biotinylated LAG3, His-tagged FGL1 protein, and an inhibitor are incubated for one hour. Then, anti-His Tb donor and dye-labeled acceptor are added and fluorescence intensity is measured using a fluorescence reader COMPONENTS: Catalog # Component Amount Storage 100330 FGL1, His tag 5 µg -80°C LAG3 (CD223), Biotin-labeled 71147 10 µg -80°C (Human) HiP™ Avoid 30017 Anti-His Tb Donor 2 x 10 µl -20°C multiple Dye-labeled Acceptor 2 x 10 µl -20°C freeze/thaw 3x FGL1 TR-FRET Buffer 4 ml -20°C cycles! Room. 79696 White, 96-well microtiter plate 1 temp MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED: Fluroescence microplate reader capable of measuring Time Resolved-Fluorescence Resonance Energy Transfer (TR-FRET) Adjustable micropipettor and sterile tips APPLICATIONS: This kit is useful for screening for inhibitors of LAG3 binding to FGL1.
    [Show full text]