CRM is a Damon Runyon-Genentech Clinical Investigator. This work was supported, in part, by grants to CRM from the Damon Runyon Cancer Research Foundation (CI-45-09), Department of Defense (W81XWH-09-2-0042), and University of North Carolina University Cancer Research Fund (UCRF). The authors wish to thank Daniel Roth for mouse husbandry assistance. The authors also wish to thank Hannah Chae, Carter McCormick, Demi Canoutas, Stephanie Gillette, and Susannah Krom for tissue culture and immunofluorescence assistance.

All animal studies were approved by the University of North Carolina Institutional Animal Care and Use Committee.
1. Culturing Cortical Astrocytes from Neonatal Mice
<ol>
	<li>Preparation
	<ol>
		<li>Crumple 2-3 delicate task tissues and place into the bottom of a flask containing 70% ethanol. Place dissecting scissors, curved forceps and 2 pairs of straight micro forceps into this flask. The tissues are used to avoid bending the micro forceps.</li>
		<li>Add 1 ml&#160;of HBSS to a 60 mm tis...

The authors have nothing to disclose.

The most critical steps to ensure proper harvest and culture of cortical astrocytes are 1) to excise the cortex without taking tissue below the corpus callosum, 2) to remove the meninges, 3) to thoroughly dissociate the cells, and 4) to enrich for GFAP+ astrocytes. Although we used mechanical (shaking) and genetic (restriction of genetic recombination with an Ad5GFAPCre vector or utilization of a dominant transforming transgene (TgGZT121) under GFAP promoter control) metho...

Astrocytomas are the most common primary brain tumor and glioblastoma (GBM), a grade IV astrocytoma, is the most common and aggressive subtype with a median survival of 12-15 months1,2. Invasion of diffuse astrocytomas, particularly GBM, precludes complete surgical resection, limits the effectiveness of adjuvant therapies, and inevitably leads to post-treatment recurrence3. Patients initially present either with de novo (primary) GBM or with lower grade astrocytomas that inevitably progresses to (secondary) GBM4. GBM is genomically heterogeneous and characterized by mutually exclusive and co-occurring mutations in genes that govern three core signaling pathways: the G1/S (Rb) cell cycle checkpoint, receptor tyrosine kinase (RTK), and TP53 pathways5-7. GBM consists of four genomic subtypes with distinct expression profiles that resemble different brain cell types, suggesting that GBM subtype is influenced by its cell of origin6,8,9. Better astrocytoma models are required to define the role of specific combinations of mutations in particular cell types during astrocytoma pathogenesis. Leveraging these models for more efficient preclinical drug development will ultimately help improve patient outcomes. Current astrocytoma models include established human cell lines, patient derived xenografts (PDX), genetically modified normal human astrocytes and neural stem cells&#160;(NSC), and genetically engineered mice (GEM)10-14. We developed an alternative, non-germline GEM (nGEM) model15 utilizing primary brain cells &#8211; cortical astrocytes and NSC &#8211; harvested from GEM harboring various combinations of floxed oncogenic alleles. The goal was to generate astrocytoma models with genetically defined cells that could be phenotypically characterized both in vitro and in vivo and potentially utilized for preclinical drug development in immune-competent mice.
Established human cell lines are the most commonly used model of astrocytoma pathogenesis and drug response in vitro and in vivo. They are technically straight forward, widely available, and have defined kinetics and tumorigenicity upon orthotopic xenografting in immunodeficient mice10,11,16-18. Their disadvantages include the inability to generate established cell lines from low-grade astrocytomas, limiting study only to high-grade astrocytomas; lack of a defined cell of origin; the presence of complex genomic abnormalities, often with genomic profiles that differ markedly from the original patient sample; and susceptibility to phenotypic and genotypic drift during serial culture in serum11,17,19-22. The phenotypic consequences of individual oncogenic mutations in established human GBM cell lines can be masked by the multitude of abnormalities that are actually present, which often precludes elucidation of direct genotype-phenotype consequences.
PDX are generated through subcutaneous passage of patient-isolated astrocytoma cells in immunodeficient mice or through their culture as non-adherent spheroids in defined, serum-free medium prior to orthotopic injection into the brains of immunodeficient mice12,23. PDX more accurately maintain the genomic landscape of human astrocytomas, but similar to established human cell lines, the phenotypic effect of individual oncogenic mutations can be masked due to their genomic complexity19,24. To define the phenotypic consequences of specific oncogenic mutations, particularly in response to novel therapies, panels of established human cell lines or PDX are frequently utilized to establish genotype-phenotype correlations, show generalizability, and minimize the likelihood of cell line-specific effects. While PDX accurately recapitulate the histopathological hallmarks of human astrocytomas, including invasion, orthotopic xenografts of established human cell lines generally do not21,23,25. Additionally, normal human astrocytes and NSC have been genetically-engineered with defined oncogenic mutations to model astrocytoma tumorigenesis in vitro and in vivo13,14,26. These cells lack the genomic complexity of established human cell lines and PDX and accurately recapitulate human astrocytoma histopathology, but require xenografting in immunodeficient rodents in vivo. Because all human cell models require immunodeficient rodent hosts to prevent immune-mediated xenograft rejection, these models fail to recapitulate the native tumor-stroma interactions of a syngeneic system and lack an intact immune system, limiting preclinical investigation of stroma-targeted and immune-modulatory therapies10,11.
GEM permit examination of the phenotypic consequences of predetermined combinations of oncogenic mutations in vivo during in situ tumorigenesis. Whereas non-conditional GEM have mutations within all tissues throughout development, conditional GEM have floxed oncogenic alleles that enable targeting of mutations by restricting Cre-mediated recombination to specific cell types through use of cell type-specific promoters10,11,15,18. Conditional astrocytoma GEM have been utilized to elucidate the functional roles of oncogenic mutations in distinct cell types within an intact brain11. The preclinical utility of in situ gliomagenesis using conditional GEM is limited by a number of factors including 1) the lack of an in vitro correlate, 2) difficulty in generating large cohorts of mice with complex genotypes, 3) long latency of in situ tumor development, 4) and stochastic tumor progression. Because in situ tumorigenesis lacks a corresponding in vitro model, drug testing in vitro cannot be performed with conventional conditional GEM models. In contrast to other cancers, conditional GEM models of astrocytomas are rarely induced by single oncogenic mutations11. Thus, complex breeding schemes are required to generate conditional GEM with multiple oncogenic mutations. Moreover, astrocytoma initiation occurs with variable penetrance after a long latency period in these models, while progression to high-grade astrocytomas generally occurs in a non-uniform, stochastic manner and ultimately gives rise to tumors with complex genomic landscapes and rapid growth kinetics27,28. The variable penetrance and stochastic nature of malignant progression in conditional GEM models requires that individual mice be screened by radiographic imaging to detect the presence and location of high-grade astrocytomas before their enrollment in preclinical drug trials. Taken together, these limitations hinder the generation and testing of the large cohorts of conditional GEM required for preclinical drug testing.
The RCAS-tva GEM system, which utilizes avian retroviral (RCAS) vectors to infect GEM engineered to express the viral receptor (tva) on specific neural cell types, has been extensively utilized to model astrocytoma tumorigenesis11. In contrast to conditional GEM, this model system enables introduction of multiple oncogenic mutations in specific cells types without the requirement for complex breeding schemes. However, it is limited by variable penetrance, the requirement for actively dividing cells to achieve viral integration, and the random insertion of transgenes into the host genome29.
Non-germline GEM (nGEM) models, which utilize cells harvested from GEM, are becoming increasingly important because they overcome many of the limitations of other model systems15. The role of initiating cell type and co-occurring mutations in astrocytoma pathogenesis are difficult to determine using established human GBM cell lines or PDX because they are derived from end-stage tumors that have accumulated extensive genetic mutations in undefined cell types during the course of malignant progression. In contrast, all grades of astrocytomas can be modeled using nGEM by inducing defined genetic mutations within specific purified brain cell types11,30. Thus, the influence of specific genetic mutations and cell type on cellular and molecular phenotypes can be determined in vitro and in vivo. Similar to established human GBM cell lines, initial in vitro drug testing using nGEM can be used to prioritize drugs for in vivo testing utilizing the same cells. Tumorigenesis in vivo can then be determined by allografting nGEM cells orthotopically into the brains of immune-competent syngeneic littermates30. These orthotopic allograft models therefore permit in vivo testing not only of conventional cytotoxic and targeted therapies, but immune-modulatory and stroma-targeted therapies as well. Finally, the role of the microenvironment on tumor initiation and progression can be determined by comparing results between nGEM and conventional GEM models using the same mutations in the same cell types.
We and others have developed astrocytoma nGEM using primary cells - astrocytes, NSC, or oligodendrocyte precursor cells (OPC) - harvested from GEM30-34. The rationale behind the development of an astrocytoma nGEM was to create a model to determine the phenotypic consequences of oncogenic mutations in specific cell types that could potentially be used for preclinical drug testing in vitro and in vivo in immune-competent animals. We harvested phenotypically WT cortical astrocytes and NSC from non-Cre expressing, conditional GEM maintained on a &#62;94% C57/Bl6 background with floxed RB pathway - Rb1loxP/loxP, or TgGZT121 &#8211; and floxed RTK/RAS/PI3K pathway - Nf1loxP/loxP, KrasG12D, PtenloxP/loxP&#160;&#8211; genes in various combinations35-39. We induced genetic recombination in vitro using adenoviral vectors encoding Cre recombinase. Because cortical astrocyte harvests contain a mixture of cell types, we used Ad5GFAPCre vectors or dominant oncogenic transgenes, such as TgGZT121 driven from the human GFAP promoter, to enrich for GFAP+ cortical astrocytes in these cultures. We defined the phenotypic consequences of G1/S (Rb), MAPK, and PI3K pathway mutations in cortical astrocytes and NSC in vitro and in vivo. MAPK and PI3K pathway-activated G1/S-defective astrocytes molecularly mimicked human proneural GBM and, upon orthotopic injection, formed tumors in a pre-defined location with uniform growth kinetics, short latencies, and the histopathological hallmarks of human GBM30. Longitudinal monitoring of tumor growth in vivo aids preclinical drug testing through normalization of treatment cohorts and quantitative analysis of tumor growth in response to treatment40. We determined tumor growth kinetics by longitudinal bioluminescence imaging of mice injected with luciferase expressing cortical astrocytes. Therefore, cortical astrocytes and NSC derived from conditional GEM provide a tractable model system for definition of functional consequences of astrocytoma-associated mutations and a potential model system for preclinical drug development.

<table border="0" cellpadding="0" cellspacing="0" width="784">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:241px;">Name of Material/ Equipment</td>
			<td style="width:191px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:233px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Dulbecco&#39;s Modified Eagle Medium (DMEM) (1X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">11995065</td>
			<td style="width:233px;">DMEM can also be purchased from other suppliers including GIBCOSigma and Cellgro</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Fetal Bovine Serum, Regular</td>
			<td style="width:191px;">Cellgro</td>
			<td style="width:119px;">35-010-CV</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Penicillin-Streptomycin</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">15140122</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Sharp-Pointed Dissecting Scissors</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">08-395</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Cartilage Thumb Forceps, Curved</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">1631</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Miltex 17-301 Style 1 Jeweler Style Forceps, Fine, 4</td>
			<td style="width:191px;">Miltex</td>
			<td style="width:119px;">17-301</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Ethanol (200 proof)</td>
			<td style="width:191px;">Decon Labs</td>
			<td style="width:119px;">2710</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Razor Blades</td>
			<td style="width:191px;">VWR</td>
			<td style="width:119px;">55411-050</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (1X), liquid</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14175-095</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">TrypLE Express (1X), Phenol Red</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">12605-010</td>
			<td style="width:233px;">Other Trypsin solutions are also suitable for cortical astrocyte havest and culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">CULTURE DISH, 60 x 15 mm</td>
			<td style="width:191px;">Thomas Scientific</td>
			<td style="width:119px;">9380H77</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">15 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352096</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">50 ml tubes</td>
			<td style="width:191px;">BD Biosciences</td>
			<td style="width:119px;">352070</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">Adenovirus stock</td>
			<td style="width:191px;">Gene Transfer Vector Core, U. Iowa</td>
			<td style="width:119px;">Ad5CMVCre</td>
			<td style="width:233px;">Store in 5 &#181;l aliquots at -80 C. Hazardous.&#160; Use Bsl2 safetyy&#160; precautions</td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium sulfate (Na2SO4)&#160;</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">238597</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Potassium sulfate (K2SO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">221325</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium chloride (MgCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M8266</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Calcium chloride (CaCl2)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">746495</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">HEPES potassium salt</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">H0527</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">D-(+)- Glucose</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">G8270&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Phenol Red</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">P3532</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Sodium hydroxide (NaOH)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5881</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Papain</td>
			<td style="width:191px;">Worthington</td>
			<td style="width:119px;">LS003127</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">L-Cysteine-HCl</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">C1276</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Syringe filter (0.22 &#181;m pore size)</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLGP033NS</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:241px;">Neurocult proliferation kit, mouse</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5702</td>
			<td style="width:233px;">This kit contains the NeuroCult NSC Basal Medium and NeuroCult NSC supplement needed for NSC culture</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">0.2% Heparin solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">7980</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PMG8041</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">bFGF&#160;</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">PHG0261</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hanks&#39; Balanced Salt Solution (HBSS) (10X)</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">14185-052</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Magnesium sulfate (MgSO4)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7506</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="26">
			<td height="26" style="height:26px;width:241px;">Sodium bicarbonate (NaHCO3)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">S5761</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">E-64</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">E3132</td>
			<td style="width:233px;">Make 10 mM stock in DMSO, store at -20 &#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">6-well plates</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">07-200-83</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Cell strainer (40 &#181;m pore size)</td>
			<td style="width:191px;">Corning</td>
			<td style="width:119px;">352340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Stem cell dissociation solution</td>
			<td style="width:191px;">Stemcell Technologies</td>
			<td style="width:119px;">5707</td>
			<td style="width:233px;">Alternatively, use gentle enzyme solutions such as Accutase</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Methyl cellulose 15 cP</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">M7027</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Dulbecco&#39;s Modified Eagle&#39;s Media 2X</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">SLM-202-B</td>
			<td style="width:233px;">For making 5% methyl cellulose solution</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">1.7mL Snap Cap Microcentrifuge Tube</td>
			<td style="width:191px;">Corning</td>
			<td align="right" style="width:119px;">3620</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Hamilton syringe, 250 &#181;l LT no needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-815-92</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">PB600-1 Antigen Dispenser</td>
			<td style="width:191px;">Hamilton</td>
			<td style="width:119px;">&#160;83700</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Disposable 18 ga needles&#160;</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9015638&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">27 ga 1/2&#34; luer tip needle</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">14-826-48</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">2,2,2-Tribromoethanol (Avertin)</td>
			<td style="width:191px;">Sigma-Aldrich</td>
			<td style="width:119px;">T48402</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Betadine</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9386574</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Puralube Opthalmic Ointment</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9689910</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">&#160;Model 900 Stereotaxic frame</td>
			<td style="width:191px;">Kopf Instruments</td>
			<td style="width:119px;"></td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">VETBOND</td>
			<td style="width:191px;">Fisher Scientific</td>
			<td style="width:119px;">NC9259532&#160;</td>
			<td style="width:233px;">Tissue adhesive&#160;</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Lidocaine&#160;</td>
			<td style="width:191px;">ShopMedVet</td>
			<td style="width:119px;">RXLIDO-EPI</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:241px;">CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS)</td>
			<td style="width:191px;">Promega</td>
			<td style="width:119px;">G3580</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">Anti-Sox2</td>
			<td style="width:191px;">Millipore</td>
			<td style="width:119px;">AB5603</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">Polyclonal Rabbit Anti-Glial Fibrillary Acidic Protein (GFAP)</td>
			<td style="width:191px;">Dako</td>
			<td style="width:119px;">Z0334&#160;</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">IVIS Kinetic</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;"></td>
			<td style="width:233px;">For in vivo imaging</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:241px;">D-Luciferin - K+ Salt Bioluminescent Substrate</td>
			<td style="width:191px;">PerkinElmer</td>
			<td style="width:119px;">122796</td>
			<td style="width:233px;">For in vivo bioluminescence imaging</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:241px;">EdU Imaging Kit</td>
			<td style="width:191px;">Invitrogen</td>
			<td style="width:119px;">C10340</td>
			<td style="width:233px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:241px;">MSCV Luciferase PGK-hygro</td>
			<td style="width:191px;">Addgene</td>
			<td style="width:119px;">18782</td>
			<td></td>
		</tr>
	</tbody>
</table>

modeling astrocytoma pathogenesis in vitro and in vivo using cortical astrocytes or neural stem cells from conditional, genetically engineered mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease pathogenesis and their utility for preclinical drug development. In order to design a better model system for these applications, phenotypically wild-type cortical astrocytes and neural stem cells (NSC) from conditional, genetically engineered mice (GEM) that harbor various combinations of floxed oncogenic alleles were harvested and grown in culture. Genetic recombination was induced in vitro using adenoviral Cre-mediated recombination, resulting in expression of mutated oncogenes and deletion of tumor suppressor genes. The phenotypic consequences of these mutations were defined by measuring proliferation, transformation, and drug response in vitro. Orthotopic allograft models, whereby transformed cells are stereotactically injected into the brains of immune-competent, syngeneic littermates, were developed to define the role of oncogenic mutations and cell type on tumorigenesis in vivo. Unlike most established human glioblastoma cell line xenografts, injection of transformed GEM-derived cortical astrocytes into the brains of immune-competent littermates produced astrocytomas, including the most aggressive subtype, glioblastoma, that recapitulated the histopathological hallmarks of human astrocytomas, including diffuse invasion of normal brain parenchyma. Bioluminescence imaging of orthotopic allografts from transformed astrocytes engineered to express luciferase was utilized to monitor in vivo tumor growth over time. Thus, astrocytoma models using astrocytes and NSC harvested from GEM with conditional oncogenic alleles provide an integrated system to study the genetics and cell biology of astrocytoma pathogenesis in vitro and in vivo and may be useful in preclinical drug development for these devastating diseases.

We developed an nGEM model system with cortical astrocytes and NSC harvested from neonatal GEM harboring floxed conditional oncogenic alleles that can be phenotypically characterized in vitro and in vivo (Figure 1). In order to investigate the consequences of oncogenic mutations specifically in cortical astrocytes in vitro, it is critical to first enrich for astrocytes. Cortical astrocyte harvests contain a mixture of microglia, astrocytes, oligodendrocytes, OPC, and neurons, b...

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Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice

Current astrocytoma models are limited in their ability to define the roles of oncogenic mutations in specific brain cell types during disease ...

modeling-astrocytoma-pathogenesis-vitro-vivo-using-cortical

Research

JoVE Journal

Neuroscience

13.3K visualizações.  University of North Carolina School of Medicine. Phenotypically wild-type astrocytes and neural stem cells harvested from mice engineered with floxed, conditional oncogenic alleles and transformed via viral Cre-mediated recombination can be used to model astrocytoma pathogenesis in vitro and in vivo by orthotopic injection of transformed cells into brains of syngeneic, immune-competent littermates.

Vídeo: Modeling Astrocytoma Pathogenesis In Vitro and In Vivo Using Cortical Astrocytes or Neural Stem Cells from Conditional, Genetically Engineered Mice