Overexpression Models: Lentiviral Modeling of Brain Cancer

Dinorah Friedmann‐Morvinski1, Oded Singer1

1 The Salk Institute for Biological Studies, La Jolla, California
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo110271
Online Posting Date:  June, 2013
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Glioblastoma multiforme (GBM) is one of the most common and most malignant of the brain tumors. Gliomas can be classified into four different grades according to their histologic characteristics; the most aggressive of the gliomas is glioblastoma multiforme (grade IV). Despite optimal treatment, the median survival is only 12 to 15 months. In the past few years, important advances were made in understanding the biology and pathology of malignant gliomas. A mouse model of brain tumors using inducible lentiviral vectors is described here. In this approach, a lenti‐vector with loxP sites flanking the gene of interest (oncogene) is injected into mice expressing Cre recombinase under the control of a brain‐specific promoter. The steps to perform cell‐type/region‐specific injection of Cre‐loxP‐controlled lentiviral vectors in the brain of adult mice are described here in detail. Curr. Protoc. Mouse Biol. 3:121‐139 © 2013 by John Wiley & Sons, Inc.

Keywords: gliomas; lentivirus gene transfer; genetic alterations; Cre‐loxP system; gene delivery

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Table of Contents

  • Introduction
  • Basic Protocol 1: Design and Cloning of Lentivectors Expressing Oncogenes and shRNAs
  • Basic Protocol 2: Production of Lentiviral Vectors
  • Basic Protocol 3: Stereotaxic Injection of Lentivirus into the Brain
  • Basic Protocol 4: Tissue Processing and Imaging
  • Commentary
  • Literature Cited
  • Figures
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Basic Protocol 1: Design and Cloning of Lentivectors Expressing Oncogenes and shRNAs

  • Primers
  • Pol III promoters plasmids (pH1, phU6 or pmU6) and appropriate lentiviral vectors (Verma Laboratory, Salk Institute for Biological Studies, )
  • Advantage GC‐2 polymerase mix (BD Biosciences) with GC melt or Taq polymerase with 7% DMSO
  • Appropriate enzymes
  • Thermal cycler
  • Additional reagents and equipment for PCR amplification (Kuslich et al., ), cloning (Finney et al., ), and sequencing (Shendure et al., )

Basic Protocol 2: Production of Lentiviral Vectors

  • 293T HEK mammalian cells (ATCC #CRL‐11268)
  • DMEM plus 10% FBS
  • 10% poly‐L‐lysine (SIGMA, cat. no. P‐4832) in PBS, filter sterilize and store indefinitely at 4°C
  • Plasmids:
    • Lentiviral transfer vector (Verma Laboraory, Salk Institute for Biological Studies, )
    • Lentiviral packaging vectors: pMDL, pRev, and pVSVG (Addgene)
    • Endotoxin‐free maxipreps (Endo‐free Maxiprep Kit, Qiagen, cat. no. 12632, or equivalent)
    • 1 µg/µl resuspended DNA
  • 2.5 M CaCl 2 stock solution: 36.75 g CaCl 2 in 70 ml ddH 2O, bring to final volume of 100 ml, dispense into 1.5‐ml microcentrifuge tubes, and store indefinitely at −20°C
  • 2× BBS solution (50 mM BES/280 mM NaCl/1.5 mM Na 2HPO 4): 16.36 g NaCl, 10.65 g BES (Calbiochem, cat. no. 391334), 0.21 g Na 2HPO 4, add ddH 2O up to 900 ml; dissolve, titrate to pH 6.95 with 1 M NaOH, bring volume up to 1 liter, filter to sterilize, and store 14‐ml aliquots indefinitely at 4°C
  • Hanks' balanced salt solution (HBSS, GIBCO, cat. no. 14175)
  • 20% sucrose in 1× HBSS (filter sterilize and store indefinitely at 4°C)
  • 12 × 15–cm tissue culture dishes
  • 50‐ml tubes
  • 37°C, 3% to 5% CO 2 tissue culture incubator
  • Fluorescent microscope
  • 37°C, 10% CO 2 tissue culture incubator
  • 0.45‐µm filter units, 500‐ml capacity (Corning, cat. no. 430773)
  • Conical bottom ultracentrifugation tubes (Beckman, cat. no. 358126)
  • Beckman ultracentrifuge and Beckman SW28 rotor (or equivalent)
  • Aspirator
  • Round‐bottom ultracentrifugation tubes (Beckman, cat. no. 326819)
  • Beckman SW55 swinging bucket rotor (or equivalent)
  • 1.5‐ml screw‐cap microcentrifuge tubes
  • 12‐well plates
CAUTION: Working with lentiviral vectors requires Biosafety level II containment. For safety procedures regarding handling of lentiviral vector preparations, see Biosafety in Microbiological and Biomedical Laboratories, 4th edition, published by the Centers for Disease Control (CDC), which can be found at http://www.cdc.gov/od/ohs/biosfty/bmb14/bmb14toc.htm.

Basic Protocol 3: Stereotaxic Injection of Lentivirus into the Brain

  • Anesthetics: 10 mg/ml ketamine and 1 mg/ml xylazine in 0.9% saline
  • 8‐ to 16‐week‐old GFAP‐Cre mice
  • Artificial tears (eye lubricant ointment, Butler, cat. no. AHS NDC 11695‐1418‐6)
  • Betadine surgical scrub
  • 70% ethanol
  • Sterile PBS
  • Hydrogen peroxide (Sigma, cat. no. H‐1009)
  • Virus solution (see protocol 2)
  • Tissue adhesive (3M Vetbond, cat. no. 1469SB)
  • Biosafety level‐2 facility
  • Warming pad
  • Electric hair trimmer or shaver
  • Stereotaxic instrument (KOPF model 900)
  • Cotton swabs
  • Surgical tools
  • Microsyringes (Hamilton, cat. no. 87925)
  • 33‐G needles for microsyringes (Hamilton, cat. no. 7762‐06)
  • Marker pen
  • Electric drill and drill burr, size no. 1 (Henry Shein, cat. no. 100‐7176)
  • 4‐0 nylon thread
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