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...

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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>
			<td style="width:267px;"></td>
		</tr>
	</tbody>
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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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Research

JoVE Journal

Medicine

12.8K ビュー.  University of Michigan School of Medicine. 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.

ビデオ: 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...

glioblastoma induction using sb mediated transposition: a technique for generating novel mouse model using transposon mediated integration of sb plasmid into neonatal mouse brain

This video describes an efficient technique to induce 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 histopathological characteristics of human...

Glioblastoma Induction Using SB Mediated Transposition: A Technique for Generating Novel Mouse Model Using Transposon Mediated Integration of SB Plasmid into Neonatal Mouse Brain

laser capture microdissection of glioma subregions for spatial and molecular characterization of intratumoral heterogeneity, oncostreams, and invasion

Laser Capture Microdissection of Glioma Subregions for Spatial and Molecular Characterization of Intratumoral Heterogeneity, Oncostreams, and Invasion

Laser microdissection (LMD) is a sensitive and highly reproducible technique that can be used to uncover pathways that mediate glioma heterogeneity and invasion. Here, we describe an optimized protocol to isolate discrete areas from glioma tissue using laser LMD followed by transcriptomic...

On the first postnatal day, perform the intraventricular injections. Prepare the injection solution by first aliquoting 20 microliters of the DNA mix containing the transposon plasmids at a final concentration of 0.5 micrograms of DNA per microliter. Next, add a 20-microliter aliquot of PEI solution to the DNA solution and let them incubate at room temperature for 20 minutes to an hour after which the mix should be stored on...

neonatal subventricular zone electroporation

Neonatal Subventricular Zone Electroporation

We demonstrate a minimally invasive technique referred to as neonatal subventricular zone electroporation. The technique consists of injecting plasmid DNA into the lateral ventricles of neonatal pups and applying electrical current to deliver and genetically manipulate neural stem...

evaluation of biomarkers in glioma by immunohistochemistry on paraffin-embedded 3d glioma neurosphere cultures

Evaluation of Biomarkers in Glioma by Immunohistochemistry on Paraffin-Embedded 3D Glioma Neurosphere Cultures

Neurospheres grown as 3D cultures constitute a powerful tool to study glioma biology. Here we present a protocol to perform immunohistochemistry while maintaining the 3D structure of glioma neurospheres through paraffin embedding. This method enables the characterization of glioma neurosphere properties such as stemness and neural...

tol2 transposon-mediated zebrafish transgenesis: a procedure to generate transgenic zebrafish following the coinjection of tol2 system into fertilized zebrafish embryos

In this video, we demonstrate a procedure to generate a stable zebrafish transgenic line constitutively expressing a fluorescent voltage indicator protein following the co-injection of the Tol2 transposon system comprising the transposase mRNA and Tol2 transposon construct encoding the fluorescent voltage...

Tol2 Transposon-Mediated Zebrafish Transgenesis: A Procedure to Generate Transgenic Zebrafish Following the Coinjection of Tol2 System Into Fertilized Zebrafish Embryos

delivery of in vivo acute intermittent hypoxia in neonatal rodents to prime subventricular zone-derived neural progenitor cell cultures

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

This article describes the methodology for administering short periods of intermittent hypoxia to postnatal day 1-8 mouse or rat pups. This approach effectively elicits a robust tissue level &#8220;priming effect&#8221; on cultured neural progenitor cells that are harvested within 30 min of hypoxia...

Delivery of In Vivo Acute Intermittent Hypoxia in Neonatal Rodents to Prime Subventricular Zone-derived Neural Progenitor Cell Cultures

determination of the optimal chromosomal location(s) for a dna element in escherichia coli using a novel transposon-mediated approach

Determination of the Optimal Chromosomal Locations for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

Here, the power of a transposon-mediated random insertion of a non-coding DNA element was used to resolve its optimal chromosomal...

Determination of the Optimal Chromosomal Location(s) for a DNA Element in Escherichia coli Using a Novel Transposon-mediated Approach

The following morning, remove the prepared injection solution from the negative 80 degrees Celsius freezer, and place it on ice. Pull the dividers in the fish breeding tanks and allow the fish to mate. In general, fish lay eggs within 20 to 30...

preparation of acute subventricular zone slices for calcium imaging

Preparation of Acute Subventricular Zone Slices for Calcium Imaging

A method to load subventricular zone (SVZ) cells with calcium indicator dyes for recording calcium activity is described.  The postnatal SVZ contains tightly packed cells including neural progenitor cells and neuroblasts. Rather than using bath loading we injected the dye by pressure inside the tissue allowing better dye...

electroporation of plasmid dna into mouse skeletal muscle

Electroporation of Plasmid DNA into Mouse Skeletal Muscle

Electroporation of plasmid DNA into skeletal muscle is a viable method to modulate gene expression without compromising muscle contractility in...

novel method of plasmid dna delivery to mouse bladder urothelium by electroporation

Novel Method of Plasmid DNA Delivery to Mouse Bladder Urothelium by Electroporation

We describe a novel method for the delivery of DNA plasmid into the urothelial cells of mouse bladder in vivo through urethra catheterization and electroporation. It offers a fast and convenient way for generating autochthonous mouse models of bladder...

delivery of dna plasmids into urothelium of mouse bladder: an electroporation-based procedure to efficiently deliver dna plasmids into urothelial cells of murine bladder to generate genetically engineered models

This video demonstrates the delivery of DNA plasmid into the urothelium of the mouse bladder using a combination of urethral catheterization and electroporation techniques. The described procedure allows the generation of mouse bladder cancer...

Delivery of DNA Plasmids into Urothelium of Mouse Bladder: An Electroporation-based Procedure to Efficiently Deliver DNA Plasmids into Urothelial Cells of Murine Bladder to Generate Genetically Engineered Models

microinjection of dna plasmid mix into the mouse brain ventricle

Microinjection of DNA Plasmid Mix into the Mouse Brain Ventricle  

isolation, expansion, and nucleofection of neural stem cells from adult murine subventricular zone

Author Spotlight: Improved Nucleofection for High-Efficiency Gene Delivery in Murine Subventricular Zone-Derived Neural Stem Cell Cultures

Here, we describe a nucleofection system designed to enhance gene delivery efficiency in expanded neural stem cells (NSCs) isolated from the adult murine subventricular zone. The findings demonstrate that this method significantly improves gene perturbation in NSCs, surpassing the effectiveness of traditional transfection protocols and enhancing cell survival...

Efficient Nucleofection of NSCs Isolated from the Murine Subventricular Zone

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the subventricular zone en-face: wholemount staining and ependymal flow

The Subventricular Zone En-face: Wholemount Staining and Ependymal Flow

The lateral ventricle walls contain the largest germinal region in the adult mammalian brain.  Traditionally, studies on neurogenesis in this region have relied on classical sectioning techniques for histological analysis.  Here we present an alternative approach, the wholemount technique, which provides a comprehensive, en-face view of this germinal...

modeling brain tumors in vivo using electroporation-based delivery of plasmid dna representing patient mutation signatures

Author Spotlight: Modeling Brain Tumors In Vivo Using Electroporation-Based Delivery of Plasmid DNA Representing Patient Mutation Signatures  

Utilizing an immunocompetent, autochthonous tumor model driven by common patient mutations for preclinical testing is critical for immunotherapeutic testing. This protocol describes a method to generate brain tumor mouse models using electroporation-based delivery of plasmid DNA that represent common patient mutations, thus providing an accurate, reproducible, and consistent mouse model.

In Vivo Brain Tumor Modeling via Electroporation-Based Delivery of Plasmid DNA

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transformation of plasmid dna into e. coli using the heat shock method

Transformation of Plasmid DNA into E. coli Using the Heat Shock Method

Transformation of plasmid DNA into E. coli using the heat shock method is a basic technique of molecular biology. It consists of inserting a foreign ...

Next, cut the muscle layer to expose the bladder. Pipette at least 20 microliters of plasmid plus Trypan Blue solution into the outer end of the catheter and attach it to the syringe. Then, inject the plasmid solution into the bladder. If the bladder turns blue, the injection is successful. Leave some liquid in the catheter to avoid creating air bubbles in the...

Use of Animal Model of Sepsis to Evaluate Novel Herbal Therapies

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several techniques, including infusion of exogenous bacterial toxin (endotoxemia) or bacteria (bacteremia), as well as surgical perforation of the cecum by cecal ligation and puncture (CLP)1-3. CLP allows bacteria spillage and fecal contamination of the peritoneal cavity, mimicking the human clinical disease of perforated appendicitis or diverticulitis. The severity of sepsis, as reflected by the eventual mortality rates, can be controlled surgically by varying the size of the needle used for cecal puncture2. In animals, CLP induces similar, biphasic hemodynamic cardiovascular, metabolic, and immunological responses as observed during the clinical course of human sepsis3. Thus, the CLP model is considered as one of the most clinically relevant models for experimental sepsis1-3.
Various animal models have been used to elucidate the intricate mechanisms underlying the pathogenesis of experimental sepsis. The lethal consequence of sepsis is attributable partly to an excessive accumulation of early cytokines (such as TNF, IL-1 and IFN-&gamma;)4-6 and late proinflammatory mediators (e.g., HMGB1)7. Compared with early proinflammatory cytokines, late-acting mediators have a wider therapeutic window for clinical applications. For instance, delayed administration of HMGB1-neutralizing antibodies beginning 24 hours after CLP, still rescued mice from lethality8,9, establishing HMGB1 as a late mediator of lethal sepsis. The discovery of HMGB1 as a late-acting mediator has initiated a new field of investigation for the development of sepsis therapies using Traditional Chinese Herbal Medicine. In this paper, we describe a procedure of CLP-induced sepsis, and its usage in screening herbal medicine for HMGB1-targeting therapies.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection, and can be simulated by a surgical technique termed cecal ligation and puncture (CLP). Here we describe a method to use CLP-induced animal model to screen medicinal herbs for therapeutic agents.

Sepsis refers to a systemic inflammatory response syndrome resulting from a microbial infection. It has been routinely simulated in animals by several ...

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome compared to matched normal tissue. Based on these analyses it is evident that there are many undiscovered genetic drivers of cancer1. Unfortunately these drivers are hidden within a much larger number of passenger anomalies in the genome that do not directly contribute to tumor formation. Another aspect of the cancer genome is that there is considerable genetic heterogeneity within similar tumor types. Each tumor can harbor different mutations that provide a selective advantage for tumor formation2. Performing an unbiased forward genetic screen in mice provides the tools to generate tumors and analyze their genetic composition, while reducing the background of passenger mutations. The Sleeping Beauty (SB) transposon system is one such method3. The SB system utilizes mobile vectors (transposons) that can be inserted throughout the genome by the transposase enzyme. Mutations are limited to a specific cell type through the use of a conditional transposase allele that is activated by Cre Recombinase. Many mouse lines exist that express Cre Recombinase in specific tissues. By crossing one of these lines to the conditional transposase allele (e.g. Lox-stop-Lox-SB11), the SB system is activated only in cells that express Cre Recombinase. The Cre Recombinase will excise a stop cassette that blocks expression of the transposase allele, thereby activating transposon mutagenesis within the designated cell type. An SB screen is initiated by breeding three strains of transgenic mice so that the experimental mice carry a conditional transposase allele, a concatamer of transposons, and a tissue-specific Cre Recombinase allele. These mice are allowed to age until tumors form and they become moribund. The mice are then necropsied and genomic DNA is isolated from the tumors. Next, the genomic DNA is subjected to linker-mediated-PCR (LM-PCR) that results in amplification of genomic loci containing an SB transposon. LM-PCR performed on a single tumor will result in hundreds of distinct amplicons representing the hundreds of genomic loci containing transposon insertions in a single tumor4. The transposon insertions in all tumors are analyzed and common insertion sites (CISs) are identified using an appropriate statistical method5. Genes within the CIS are highly likely to be oncogenes or tumor suppressor genes, and are considered candidate cancer genes. The advantages of using the SB system to identify candidate cancer genes are: 1) the transposon can easily be located in the genome because its sequence is known, 2) transposition can be directed to almost any cell type and 3) the transposon is capable of introducing both gain- and loss-of-function mutations6. The following protocol describes how to devise and execute a forward genetic screen using the SB transposon system to identify candidate cancer genes (Figure 1).

Identification of Sleeping Beauty Transposon Insertions in Solid Tumors using Linker-mediated PCR

A method of identifying unknown drivers of carcinogenesis using an unbiased approach is described. The method uses the Sleeping Beauty transposon as a random mutagen directed to specific tissues. Genomic mapping of transposon insertions that drive tumor formation identifies novel oncogenes and tumor suppressor genes

Genomic, proteomic, transcriptomic, and epigenomic analyses of human tumors indicate that there are thousands of anomalies within each cancer genome ...

Image-guided Convection-enhanced Delivery into Agarose Gel Models of the Brain

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows for positive pressure bulk flow to deliver greater quantities of therapeutics to an intracranial target than traditional drug delivery methods. The clinical utility of real time MRI guided CED (rCED) lies in the ability to accurately target, monitor therapy, and identify complications. With training, rCED is efficient and complications may be minimized. The agarose gel model of the brain provides an accessible tool for CED testing, research, and training. Simulated brain rCED allows practice of the mock surgery while also providing visual feedback of the infusion. Analysis of infusion allows for calculation of the distribution fraction (Vd/Vi) allowing the trainee to verify the similarity of the model as compared to human brain tissue. This article describes our agarose gel brain phantom and outlines important metrics during a CED infusion and analysis protocols while addressing common pitfalls faced during CED infusion for the treatment of neurological disease.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. In order to prepare health care professionals for adoption of CED, accessible training models are needed. We describe the use of agarose gel as such a model of the human brain for testing, research, and training.

Convection-enhanced delivery (CED) has been proposed as a treatment option for a wide range of neurological diseases. Neuroinfusion catheter CED allows ...

Orthotopic Injection of Breast Cancer Cells into the Mammary Fat Pad of Mice to Study Tumor Growth.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the functions of proteins involved in oncogenic progression or help to discover new tumor suppressors. In addition, injecting cancer cells into mice with different genotypes might provide a better understanding of the importance of the stromal compartment. Many models may be useful to investigate certain aspects of disease progression but do not recapitulate the entire cancerous process. In contrast, breast cancer cells engraftment to the mammary fat pad of mice better recapitulates the location of the disease and presence of the proper stromal compartment and therefore better mimics human cancerous disease. In this article, we describe how to implant breast cancer cells into mice orthotopically and explain how to collect tissues to analyse the tumor milieu and metastasis to distant organs. Using this model, many aspects (growth, angiogenesis, and metastasis) of cancer can be investigated simply by providing a proper environment for tumor cells to grow.

Cancer is a complex disease that is influenced by the tissue surrounding the tumor as well as local pro- and anti-inflammatory mediators. Therefore, orthotropic injection models, rather than subcutaneous models may be useful to study cancer progression in a manner that better mimics human pathology.

Breast cancer growth can be studied in mice using a plethora of models. Genetic manipulation of breast cancer cells may provide insights into the ...

Mouse Kidney Transplantation: Models of Allograft Rejection

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely replicates both the technical and pathological processes that occur in human renal transplantation. Although mouse models of allogeneic rejection in organs other than the kidney exist, and are more technically feasible, there is evidence that different organs elicit disparate rejection modes and dynamics, for instance the time course of rejection in cardiac and renal allograft differs significantly in certain strain combinations. This model is an attractive tool for many reasons despite its technical challenges. As inbred mouse strain haplotypes are well characterized it is possible to choose donor and recipient combinations to model acute allograft rejection by transplanting across MHC class I and II loci. Conversely by transplanting between strains with similar haplotypes a chronic process can be elicited were the allograft kidney develops interstitial fibrosis and tubular atrophy. We have modified the surgical technique to reduce operating time and improve ease of surgery, however a learning curve still needs to be overcome in order to faithfully replicate the model. This study will provide key points in the surgical procedure and aid the process of establishing this technique.

Here, we present a protocol to study the immunology of rejection. The surgical model presented reports a short operating time and a concise technique. Depending on the donor-recipient strain combination, the transplanted kidney may develop acute cellular rejection or chronic allograft damage, defined by interstitial fibrosis and tubular atrophy.

Rejection of the transplanted kidney in humans is still a major cause of morbidity and mortality. The mouse model of renal transplantation closely ...

A Piglet Model of Neonatal Hypoxic-Ischemic Encephalopathy

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the world&#8217;s 2.9 million neonatal deaths. Animal models of HIE have contributed to the understanding of the pathophysiology in HIE, and have highlighted the dynamic process that occur in brain injury due to perinatal asphyxia. Thus, animal studies have suggested a time-window for post-insult treatment strategies. Hypothermia has been tested as a treatment for HIE in pdiglet models and subsequently proven effective in clinical trials. Variations of the model have been applied in the study of adjunctive neuroprotective methods and piglet studies of xenon and melatonin have led to clinical phase I and II trials1,2. The piglet HIE model is further used for neonatal resuscitation- and hemodynamic studies as well as in investigations of cerebral hypoxia on a cellular level. However, it is a technically challenging model and variations in the protocol may result in either too mild or too severe brain injury. In this article, we demonstrate the technical procedures necessary for establishing a stable piglet model of neonatal HIE. First, the newborn piglet (&#60; 24 hr old, median weight 1500 g) is anesthetized, intubated, and monitored in a setup comparable to that found in a neonatal intensive care unit. Global hypoxia-ischemia is induced by lowering the inspiratory oxygen fraction to achieve global hypoxia, ischemia through hypotension and a flat trace amplitude integrated EEG (aEEG) indicative of cerebral hypoxia. Survival is promoted by adjusting oxygenation according to the aEEG response and blood pressure. Brain injury is quantified by histopathology and magnetic resonance imaging after 72 hr.

Hypoxic-ischemic encephalopathy following perinatal asphyxia can be studied using animal models. We demonstrate the procedures necessary for establishing a piglet model of neonatal hypoxic-ischemic encephalopathy.

Birth asphyxia, which causes hypoxic-ischemic encephalopathy (HIE), accounts for 0.66 million deaths worldwide each year, about a quarter of the ...

Surgical Models of Gastroesophageal Reflux with Mice

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to induce reflux of gastric contents, duodenal contents or mixed contents. Surgical procedures and general principles have been described in detail. A researcher with surgical experience should be able to grasp the technique after a short period of practice. After surgery, most mice can survive and develop reflux esophagitis similar to that in humans. However, it should be noted that histological differences between mouse and human esophagus are the inherent limitations of these surgical models. If used for research on Barrett&#8217;s esophagus and adenocarcinoma, these procedures may need to be combined with genetic modifications.

This article demonstrates surgical procedures of gastroesophageal reflux with mice. These models are useful tools for research on mechanisms and treatment of gastroesophageal reflux disease and potentially Barrett&#8217;s esophagus and esophageal adenocarcinoma.

Multiple surgical procedures have been reported to induce gastroesophageal reflux in animals. Herein, we report three surgical models with mice aiming to ...

Myocardial Infarction in Neonatal Mice, A Model of Cardiac Regeneration

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and regeneration, and to define new targets for therapeutics. For decades, models of complete heart regeneration existed in amphibians and fish, but a mammalian counterpart was not available. The recent discovery of a postnatal window during which mice possess regenerative capabilities has led to the establishment of a mammalian model of cardiac regeneration. A surgical model of mammalian cardiac regeneration in the neonatal mouse is presented herein. Briefly, postnatal day 1 (P1) mice are anesthetized by isoflurane and placed on an ice pad to induce hypothermia. After the chest is opened, and the left anterior descending coronary artery (LAD) is visualized, a suture is placed around the LAD to inflict myocardial ischemia in the left ventricle. The surgical procedure takes 10-15 min. Visualizing the coronary artery is crucial for accurate suture placement and reproducibility. Myocardial infarction and cardiac dysfunction are confirmed by triphenyl-tetrazolium chloride (TTC) staining and echocardiography, respectively. Complete regeneration 21 days post myocardial infarction is verified by histology. This protocol can be used to as a tool to elucidate mechanisms of mammalian cardiac regeneration after myocardial infarction.

This protocol describes a highly reproducible model of cardiac regeneration by surgical induction of myocardial infarction in the left ventricle of postnatal day 1 mice. The method involves induction of hypothermic anesthesia and ligation of the left anterior descending coronary artery.

Myocardial infarction induced by coronary artery ligation has been used in many animal models as a tool to study the mechanisms of cardiac repair and ...

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to numerous external influences including antibiotics use and diet, which impacts susceptibility to autoimmune and inflammatory diseases. Disorders such as Inflammatory Bowel Disease (IBD) are characterized by a massive influx of immune cells to the intestines. However, immune cells conditioned by the microbiota may additionally emigrate out of the intestines to influence immune responses at extra-intestinal sites. Thus, there is a need to identify and characterize cells that may carry microbial messages from the intestines to distal sites. Here, we describe a method to label cells in the colon of newborn mice in vivo that enables their identification at extra-intestinal sites after migration.

In Vivo Photolabeling of Cells in the Colon to Assess Migratory Potential of Hematopoietic Cells in Neonatal Mice

The protocol described here utilizes a photolabeling approach in newborn mice to specifically identify immune cells that emigrate from the colon to extra-intestinal sites. This strategy will be useful to study host-microbiome interactions in early life.

Enteric bacterial communities are established early in life and influence immune cell development and function. The neonatal microbiota is susceptible to ...

Full-Endoscopic Isolation Zone Technique for the Treatment of Lumbar Disc Herniation

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its clinical symptoms often include severe low back pain, limited lumbar movement, sciatic nerve pain in the lower limbs, and even cauda equina syndrome. The common treatment for LDH is a conservative treatment scheme involving medicine, rest, and physical therapy. However, if the conservative treatment scheme is ineffective, a surgical treatment approach is adopted. Traditional open lumbar surgery has some disadvantages, including the potential for severe surgical trauma, severe blood loss during the operation, instability of the lumbar spine, and loss of the lumbar motor unit. Among the minimally invasive surgical schemes, full-endoscopic spine surgery (FESS) is undoubtedly the most appropriate and has the advantages of minimal trauma, high safety, quick postoperative recovery, and the retention of the stable structure and the motor unit of the lumbar spine. However, simultaneously, incomplete removal of the nucleus pulposus and residual nerve dysfunction after surgery can occur. To avoid these shortcomings, we studied a specific spinal endoscopy technique, the "isolation zone" surgical strategy, which can effectively block the pain from the nerve conduction pathway by completely relieving the nerve compression and nerve dysfunction through the orderly treatment of the protruding nucleus pulposus, the fissure of the annulus fibrosus, the sinus nerve, and the surrounding inflammatory soft tissues.

Here, we present a protocol for the &#34;isolation zone&#34; technique in the treatment of lumbar disc herniation (LDH) under full-endoscopic spine surgery (FESS), including intervertebral foramen formation, targeted catheterization, nucleus pulposus resection, and annulus fibrosus formation, which together completely block the pain from the nerve conduction pathway.

Lumbar disc herniation (LDH) is a type of serious sinus or sciatic nerve dysfunction caused by nucleus pulposus protrusion and annulus fibrosus tears. Its ...