Overexpression Models: Lentiviral Modeling of Brain Cancer
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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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PDF or HTML at Wiley Online LibraryTable 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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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Design and Cloning of Lentivectors Expressing Oncogenes and shRNAs
Materials
Basic Protocol 2: Production of Lentiviral Vectors
Materials
Basic Protocol 3: Stereotaxic Injection of Lentivirus into the Brain
Materials
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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. View Image -
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. View Image -
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. View Image -
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×. View Image -
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. View Image
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