Name: transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma
Uploaded: 2015-02-22T14:00:00
Description: Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

We thank Dr. John Ohlfest and Dr. Stacey Decker13 for the generous gift of plasmids and for the training provided to master this method. We thank Marta Dzaman for help with standardizing the Hematoxylin-Eosin staining procedure of paraffin-embedded sections. We also thank Molly Dahlgren for perusing this manuscript and providing helpful suggestions. This work is supported by NIH/NINDS grants to MGC and PRL, Leah&#8217;s Happy Hearts Foundation grant to MCG and PRL. Alex&#8217;s Lemonade Foundation young Investigator Award and the St. Baldrick&#8217;s Foundation Fellowship to CK.

NOTE: All animal protocols have been approved by the University of Michigan Committee for the Use and Care of Animals (UCUCA).
1. Cloning of the Sequence of Interest into New Sleeping Beauty Transposons
NOTE: To insert genes or inhibitory elements (as shRNA) using the Sleeping Beauty transposase system, clone the sequence of interest into the backbone of a pKT or pT2 plasmid. Direct the cloning such that the regulatory elements, gene of interest and markers remain fla...

<ol>
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In this article, we detail a versatile and reproducible method for generating new models of GBM using SB transposase- mediated integration of oncogenic plasmid DNA into cells surrounding the subventricular zone of neonatal mice. We present a protocol to generate transposon plasmids with new genes of interest, illustrate how to monitor the progression of the tumors in live animals, and how to characterize histo-pathological and immuno-histochemical features of these tumors.
As our lab <...

Glioblastoma multiforme (GBM) is the most common (60%) primary brain tumor in adults, with a median survival of 15-21 months when treated with surgery, radiation therapy, and chemotherapy1. Novel therapies for GBM are imperative. Experimental therapies require testing in animal models which adequately reproduce the salient features of the human disease. Strategies to induce GBM in rodents include chemical mutagenesis with alkylating agents, germline or somatic genetic alterations, or transplantation using glioma cell lines2. The most commonly used models employ the implantation of glioma cell lines, either orthotopically, into the brain or subcutaneously using syngeneic cells in animals with identical genotype; or xenogeneic cells, most commonly human GBM cell lines, implanted in immune compromised mice3. Xenografts offer many advantages for the study of intracranial tumors: convenience of reproducibility, standardized growth rates, time of death and tumor localization. However, these models have limitations due to the artificial, invasive surgical approach used for implantations and limited ability to accurately reproduce histological features characteristic of human GBM (WHO grade IV): pseudo-pallisading necrosis, nuclear atypias, diffuse invasion, micro-vascular proliferation and the formation of glomeruloid vascular abnormalities4-7. Induction of GBM by altering the genome of somatic cells with oncogenic DNA, either with viral vectors8-12, or with transposon-mediated integration13, reproduces more closely the etiology of human disease and recapitulates histo-pathological features of human GBM.
Historically, tumors of the CNS have been classified based on the perceived cell of origin, which it was observed, would be a predictive factor for survival14. GBMs are classified into primary and secondary. Emerging evidence today points to the highly heterogeneous nature of primary glioblastoma15 . Secondary GBM (15%), the result of malignant transformation of low grade astrocytomas (WHO grade I) and anaplasic astrocytomas (WHO grade II), are associated with earlier onset of the disease, better prognosis and a &#8220;proneural&#8221; pattern of gene expression, whereas primary GBM (85%) show a late onset, poor prognosis and glial (classical), neural or mesenchymal expression patterns. Whether these patterns of gene expression correlate with the actual cell of origin of the tumor is still being actively investigated. Accumulating data shows that the combination of genetic mutations associated with GBMs are predictive for survival. For example, loss of heterozygocity (LOH) of chromosomes 1p/19q, IDH1 mutations, PDGFR&#945; amplifications, are associated with secondary GBMs, proneural expression pattern and better prognosis, whereas EGFR overexpression, Notch and Sonic hedgehog pathway activation, Nf1 and PTEN loss and mutations of p53 are correlated with neural, classical, or mesenchymal primary GBM and worse prognosis16,17. The advent of large scale sequencing projects and the accumulation of numerous patient specimens available for testing brings a wealth of new information with respect to genetic mutations and pathways implicated in GBM pathogenesis and progression and the possibility of individualized medicine, where therapies can be specifically tailored to the genetic abnormalities of the patient. Ultimately, to assess the predictive value of these mutations and pathways in a systematic way, and to test possible treatments in each case, requires animal models of GBM with pre-determined genetic alterations. Transposon mediated integration of genomic DNA offers a feasible approach.
The Sleeping Beauty transposon system, member of the Tc1/mariner class of transposons, was &#8220;awakened&#8221; (constructed) in a multi-step process of site specific mutagenesis from a salmonoid transposase gene, which became dormant more than 10 million years ago18,19. In essence, DNA transposons flanked by specific sequences (inverted repeats/direct repeats: IR/DR) can be integrated into the genome in a &#8220;cut and paste&#8221; manner by means of the activity of the Sleeping Beauty transposase. The transposase recognizes the ends of the IR sites, excises the transposon and integrates it randomly into another DNA site between the bases T and A, bases which are duplicated at each end of the transposon during transposition (Figure 1a). The Sleeping Beauty transposase is comprised of three domains, a transposon binding domain, a nuclear localization sequence and a catalytic domain. Four transposase molecules are required to bring the two ends of the transposon together and allow for transposition, however, if too many molecules of transposase are present, they can dimerize and tetramerize to inhibit the transposition reaction20. An efficient transposition reaction requires an optimal ratio of transposase to transposons. The DNA encoding the transposase can be delivered on the same plasmid with the transposon (in cis) or on a different plasmid (in trans). To ensure the optimal ratio between the transposase and the transposons, a promoter with adequate activity can be chosen for the expression of the transposase (for the &#8220;cis&#8221; model) or the ratio of the plasmids in the injection solution can be optimized (for the &#8220;trans&#8221; model). The Sleeping Beauty transposon system can be used successfully for functional genomics, insertional mutagenesis, transgenesis and somatic gene therapy21. Being a synthetic construct re-engineered to a functional molecule from a dormant salmonoid variant, the Sleeping Beauty transposase does not bind to other transposons in humans or other mammals20. Since its discovery, molecular engineering has enhanced the transposition efficacy of the SB transposon system through changes in the IR sequences and addition of TATA dinucleotides flanking the transposon, resulting in the pT2 transposons. These transposons have optimized binding of the SB to the IR site and increased efficacy of excision. The SB transposase also underwent significant improvement; the transposase used in experiments presented herein is the SB100X, a hyperactive transposase generated by a DNA shuffling strategy followed by a large-scale genetic screen in mammalian cells22.
In this report we present a rapid, versatile and reproducible method to induce intrinsic GBM in mice with non-viral, transposon-mediated integration of plasmid DNA into cells of the sub-ventricular zone of neonatal mice23. We present a simple way to create transposons with novel genes amenable for genomic integration using the Sleeping Beauty transposase and demonstrate how to monitor plasmid uptake and disease progression using bioluminescence. We also characterize histological and immuno-histochemical features of the GBM reproduced with this model. In addition, we present a quick method to generate primary GBM cell lines from these tumors. The Sleeping Beauty model, in which tumors are induced from cells original to the animal, allows the functional assessment of the role of candidate GBM genes in the induction and progression of tumors. This system is also well suited for the testing of novel GBM treatments, including immune therapies in immuno-competent mice, without the need of invasive inflammatory surgical procedures, which may alter the local microenvironment.

<table border="0" cellpadding="0" cellspacing="0" width="952">
	<tbody>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Name of Material/ Equipment</td>
			<td style="width:199px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:267px;">Comments/Description</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Stoelting&#39;s Lab Standard with Rat and Mouse Adaptors</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">51670</td>
			<td style="width:267px;">Other companies produce similar frames, any of them with small mouse adaptors are suitable&#160;</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">Quintessential Stereotaxic Injector (QSI)</td>
			<td style="width:199px;">Stoelting</td>
			<td style="width:119px;">53311</td>
			<td style="width:267px;">Injections can be made without it, ,but&#160;&#160;&#160; the automatic injector allows for increased reproducibility and convenience</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:367px;">10 &#956;L 700 series hand fitted MICROLITER syringe</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">Model 701</td>
			<td style="width:267px;">This syringe model will deliver from 0.5 to 500 ul of solution reliably. Other syrnges (fro example 5 ul, Model 75) may also be used</td>
		</tr>
		<tr height="120">
			<td height="120" style="height:120px;width:367px;">30 gauge gauge hypodermic needle 
			(1.25&#34;, 15o bevel)&#160;</td>
			<td style="width:199px;">Hamilton</td>
			<td style="width:119px;">S/O# 197462</td>
			<td style="width:267px;">This needle is ideal to pierce the skin and the skull of a neonatal mouse without the need of other invasive procedures. If it gets dull (doesn&#39;t easily enter the skin), it needs to be replaced.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">in vivo-jetPEI</td>
			<td style="width:199px;">Polyplus Transfection</td>
			<td style="width:119px;">201-10G</td>
			<td style="width:267px;">Aliquot in small volumes and keep at -20oC&#160;</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">D-Luciferin, Potassium Salt</td>
			<td style="width:199px;">Goldbio.com</td>
			<td style="width:119px;">LuckK-1g</td>
			<td style="width:267px;">Other sources of firefly lucifierase are just as adequate</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Hematoxylin Solution, Harris Modified</td>
			<td style="width:199px;">Sigma Aldrich</td>
			<td style="width:119px;">HHS128-4L</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:367px;">Tissue-Tek O.C.T. Compound</td>
			<td style="width:199px;">Electron Microscopy Sciences</td>
			<td style="width:119px;">62550-01</td>
			<td style="width:267px;">for embedding brains for cryosectioning and immunohistovhemistry</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Advanced DMEM/F-12, no glutamine</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">12634-010</td>
			<td style="width:267px;">for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">B-27&#168; Supplement (50X), serum free&#160;</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17504-044</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">N2 Supplement (100x)</td>
			<td style="width:199px;">Life Technologies</td>
			<td style="width:119px;">17502-048</td>
			<td style="width:267px;">serum free supplement for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Normocyn</td>
			<td style="width:199px;">InvivoGen</td>
			<td style="width:119px;">ant-nr-1</td>
			<td style="width:267px;">anti-mycoplasma agent for the culture of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human EGF</td>
			<td style="width:199px;">PEPROTECH</td>
			<td style="width:119px;">AF-100-15</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Recombinant Human FGF-basic</td>
			<td style="width:199px;">PEPROTECH</td>
			<td>100-18B</td>
			<td style="width:267px;">growth factor supplement for the culture of&#160; neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">HyClone HyQTase&#160; Cell Detachment Reagent&#160;</td>
			<td style="width:199px;">Thermo Scientific</td>
			<td style="width:119px;">SV3003001</td>
			<td style="width:267px;">for the dissociation of neurospheres</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;">Kimble-Chase Kontes Pellet Pestle</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>K749510-0590</td>
			<td style="width:267px;">for the dissociation of freshly dissected tumors</td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;">Falcon Cell Strainers mesh size 70um</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td>08-771-2</td>
			<td style="width:267px;">for generating single cell suspensions of dissociated neurospheres</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Xylenes Histological grade</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">C8H10</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:367px;">Protocol Harris Hematoxylin Mercury free ( acidified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">245-678</td>
			<td style="width:267px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:367px;">Protocol Eosyn Y Solution ( intensified)</td>
			<td style="width:199px;">Fisher Scientific</td>
			<td style="width:119px;">314-631</td>
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transposon mediated integration of plasmid dna into the subventricular zone of neonatal mice to generate novel models of glioblastoma

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary brain tumor in adults with dismal prognosis. New potential therapies are rapidly emerging from the bench and require systematic testing in experimental models which closely reproduce the salient features of the human disease. Herein we describe in detail a method to induce new models of GBM with transposon-mediated integration of plasmid DNA into cells of the subventricular zone of neonatal mice. We present a simple way to clone new transposons amenable for genomic integration using the Sleeping Beauty transposon system and illustrate how to monitor plasmid uptake and disease progression using bioluminescence, histology and immuno-histochemistry. We also describe a method to create new primary GBM cell lines. Ideally, this report will allow further dissemination of the Sleeping Beauty transposon system among brain tumor researchers, leading to an in depth understanding of GBM pathogenesis and progression and to the timely design and testing of effective therapies for patients.

To characterize histo-pathological features of SB-induced glioblastomas, C57/ BL6 neonatal mice were injected at P1 with a plasmid encoding luciferase (pT2/SB100x-Luc) in combination with plasmids encoding transposons with oncogenic DNA, i.e., NRAS (pT/CAGGS-NRASV12) and SV40 LgT (pT/CMVSV40-LgT) (Figure 3c) or a plasmid encoding a short hairpin p53 with PDGF&#946; and a GFP reporter (pT2shp53/GFP4/mPDGF&#946;) in combination with NRAS (Figure 3d). Animals were monitored for bio...

Watch this Scientific Journal Video about Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma at JoVE.com

Transposon Mediated Integration of Plasmid DNA into the Subventricular Zone of Neonatal Mice to Generate Novel Models of Glioblastoma

Here we describe an efficient and versatile protocol to induce, monitor and analyze novel glioblastomas (GBM) using transposon DNA injected into the ventricles of neonatal mice. Cells of the subventricular zone, which take up the plasmid, transform, proliferate and generate tumors with histo-pathological characteristics of human GBM.

An urgent need exists to test the contribution of new genes to the pathogenesis and progression of human glioblastomas (GBM), the most common primary ...

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Medicine

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