Overexpression Models: Lentiviral Modeling of Brain Cancer

互联网2013-12-31

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Abstract
Table of Contents
Materials
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Literature Cited

Abstract

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 lox P 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?lox P?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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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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Materials

Basic Protocol 1: Design and Cloning of Lentivectors Expressing Oncogenes and shRNAs

Materials

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

Materials

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 CaCl₂ stock solution: 36.75 g CaCl₂ in 70 ml ddH₂ O, 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 Na₂ HPO₄ ): 16.36 g NaCl, 10.65 g BES (Calbiochem, cat. no. 391334), 0.21 g Na₂ HPO₄ , add ddH₂ O 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% CO₂ tissue culture incubator
Fluorescent microscope
37°C, 10% CO₂ 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, 4^th 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

Materials

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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Figures

Figure 1. Flow chart summarizing the steps to induce glioma tumors by lentivirus injection into adult mouse brain. The first step consists of designing and cloning the desired oncogene/tumor suppressor genes in the lentiviral vector backbone. Once the lentivector has been validated in vitro, the next step will be to transfect 293T cells to prepare high‐titer lentivirus (biological titer is confirmed by flow cytometry analysis). The concentrated lentivirus is stereotaxically injected into the brain of CRE‐transgenic mice to induce the glioma tumors. Finally, the obtained tumors can be analyzed by different imaging techniques such as immunohistochemistry, immunofluorescence, or biolumminiscence imaging in vivo. GFP, green fluorescent protein; IRES, internal ribosome entry site; RFP, red fluorescent protein; CMV, cytomegalovirus.

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Figure 2. (A ) Schematic representation of the lentiviral vector. The upper two schema show the basic pTomo mock construct (Marumoto et al., ) and the lower two schema show the H‐RasV12‐shp53 lentivector described. The floxed RFP fragment keeps the translation of the H‐RasV12 in an “off” state. Only in the presence of Cre recombinase, the floxed RFP cassette is cut out resulting in the expression of the H‐RasV12 gene. The shRNA under the H1 promoter targets the p53 protein. (B ) A western blot showing expression of Flag H‐RasV12 expression induced by Cre recombinase and silencing of p53. In the left‐side blot, MEF (mouse embryonic fibroblasts) Ikk2−/− were infected with mock virus (lane 1), H‐RasV12‐shp53 (1 and 2 indicate different clones) plus Cre‐expressing lentivirus (LVs) (lanes 2 and 3), and without (w/o) Cre LV (lane 4). Cell lysates were processed for western blot using anti‐Flag antibody. For the right‐side blot, MEF Ikk2−/− cells were infected with mock virus (lane 1), H‐RasV12‐shp53 plus Cre (lane 2), and in lane 3 a positive control for p53 was run. Tubulin detection was used as loading control. (C ) Immunofluorescent microscopy showing H‐RasV12 expression (GFP) induced by Cre recombinase. Primary astrocytes isolated from GFAP‐Cre mice (which express Cre) were infected with H‐RasV12‐shp53. The infected cells express GFP but lost expression of RFP. Blue = Dapi (nuclear staining); green = GFP.

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Figure 3. Diagram showing the position of the mouse head on the stereotaxic instrument and the specific coordinates for injection of the virus in different locations of the brain. AP = anterior/posterior, lat = lateral, vent = ventral, HP = hippocampus, CTX = cortex, SVZ = subventricular zone.

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Figure 4. Brain tumors induced by H‐RasV12‐shp53 lentivirus injected in GFAP‐Cre mice. (A ) Images of GFAP‐Cre mice showing tumor formation (enlarged head). (B ) Hematoxilin and eosin (H&E) staining showing the common features of glioblastomas: (1) hemorrhage (magnification 4×), (2) necrotic areas surrounded by high density cellular regions (magnification 10×), (3) perivascular infiltration and multinucleated giant cells (arrows; magnification 40×), and (4) pseudopalisading (magnification 10×). (C ) Representative images of immunohistochemical analysis using the indicated antibodies. Glial fibrilary acidic protein (GFAP). Magnification 40×.

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Figure 5. (A ) Representative confocal microscopy images of 40‐µm tumor sections labeled with the indicated antibodies. Magnification 20×. (B ) Bioluminiscent image (BLI) of luciferase‐label tumor bearing GFAP‐Cre mouse (red arrow and circle points to the tumor). The GFAP‐Cre mouse was injected with H‐RasV12‐shp53 (luciferase) into the hippocampus and 8 weeks later the tumor was visualized by BLI.

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Literature Cited

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Overexpression Models: Lentiviral Modeling of Brain Cancer

Abstract

Table of Contents

Materials

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

Figures

Videos

Literature Cited