Name: generation of tumor organoids from genetically engineered mouse models of prostate cancer
Uploaded: 2019-06-13T14:00:00
Description: Watch this Scientific Journal Video about Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer at JoVE.com

The authors would like to thank the Calvin Kuo Laboratory at Stanford University for providing HEK293 cells stably transfected with either HA-mouse Noggin-Fc or HA-mouse Rspo1-Fc. We would also like to thank Dr. Dean Tang for allowing us access the fluorescent dissection microscope in his laboratory. This work was supported by CA179907 to D.W.G. from the National Cancer Institute. Shared resources at Roswell Park Comprehensive Cancer Center were supported by National Institutes of Health Cancer Center Support Grant CA016056.

Animal procedures described here were performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at the Department of Laboratory Animal Resources, Roswell Park Comprehensive Cancer Center, Buffalo, New York.
NOTE: Male mice to be dissected to isolate prostates or prostate tumors for generation of organoids should have at least reached the age of sexual maturity &#8212; about 8-10 weeks of age. Specific ages of mice can vary amongst studies. Some fac...

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Critical steps within the protocol for prostate tumor dissection and organoid generation 
Removal of non-prostate tissue and fine dissection of the mouse prostate tumor is crucial for the optimal generation of cancer organoids since both non-prostate epithelial cells and normal prostate epithelial cells will generate organoids. For solid prostate tumors specifically, it is crucial to isolate areas of viable tumor to remove contamination with necrotic tissue that would reduce the number of viable cel...

Since the late 1980s, the ability to alter genes by homologous recombination has greatly advanced the study of biological systems1. Inducible, tissue-, or cell-specific promotor systems and site-specific recombinases, such as Cre-loxP, has advanced genetic studies by facilitating control over genetic modifications both temporally and spatially2,3,4. The combination of these genetic strategies has created a wide array of experimental model systems5,6,7.
Genetically engineered mouse models (GEMMs) are an integral tool to assess how individual genes or groups of genes affect mammalian development and disease. In pre-clinical cancer research, GEMMs are the most physiologically-relevant and rigorous method to study cancer development, progression, and treatment8. Our laboratory specializes in generating and characterizing cancer GEMMs.
The most highly diagnosed non-cutaneous cancer among men in the United States is prostate cancer (PCa). The majority of patients with PCa have low-risk disease and high likelihood of survival, but survival rates decline drastically when disease is diagnosed at advanced stages or if targeted hormonal therapy induces progression to aggressive, non-curable PCa subtypes9,10. Our laboratory has developed GEMMs that utilize floxed alleles of one or more tumor suppressor genes. Recombination and loss of tumor suppressor gene expression occurs specifically in the prostate because we have introduced a transgene with Cre recombinase downstream of the probasin promoter activated only in prostate epithelial cells11,12. We have also bred our GEMMs to contain a Cre reporter transgene called mT/mG, which induces Tomato fluorescent protein expression in cells lacking Cre and green fluorescent protein (GFP) expression in cells with Cre13. While the presentation of this method and our representative results show GEMMs we study in our laboratory, this protocol can be used to generate prostate cancer organoids from any mouse model. However, as discussed in detail in our representative results section, we have observed that certain tumor characteristics are optimal for prostate cancer organoid generation.
In the last decade, new methods of culturing cells from tissues of epithelial origin has led to significant advances in our ability to model organ systems in vitro14,15. The term &#34;3D cell culture&#34;&#160;has been attributed to the techniques involved in establishing and maintaining organoids, which can be generally defined as structures made up of cells that assemble secondary architecture driven by organ-specific cell lineage characteristics16. These new methods are distinct from classic 2D cell culture in that cells do not require transformation or immortalization for long term growth; thus, 3D cultures of normal cells can be compared to diseased cells. This is particularly valuable in cancer research where normal cell control cultures have typically not been available. In addition, organoids spontaneously form secondary tissue architectures with appropriately differentiated cell types, making them a better model system to understand cancer in vitro than 2D cell lines17. Our laboratory has created 3D organoid lines from tumor issue isolated from our PCa GEMMs to complement our in vivo data and perform experiments which would not be feasible in GEMMs.&#160;
In this article, we present written and visual protocols for the complete necropsy of PCa GEMMs, including dissection of distinct mouse prostate lobes and metastatic lesions. We describe and show a step-by-step method for generating organoids from mouse prostate tumors based on a protocol previously published by Drost et al. for deriving organoids from normal mouse prostate epithelial tissue18.

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			<td height="21" style="height:21px;width:217px;">0.25 % Trypsin+2.21 mM EDTA</td>
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			<td height="42" style="height:42px;width:217px;">1 1/4 in, 23 gauge, disposable syringe needles</td>
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			<td height="21" style="height:21px;width:217px;">Advanced DMEM/F12+++</td>
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			<td height="21" style="height:21px;width:217px;">HEPES (1M)</td>
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			<td height="42" style="height:42px;width:217px;">human recombinant Epidermal growth factor (EGF)</td>
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			<td>AF-100-15</td>
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			<td height="21" style="height:21px;width:217px;">L-glutamine (200 mM)</td>
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			<td height="21" style="height:21px;width:217px;">N-Acetyl-L-Cysteine</td>
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			<td height="21" style="height:21px;width:217px;">Penicillin-Streptomycin</td>
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			<td height="21" style="height:21px;width:217px;">Precision balance</td>
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			<td height="42" style="height:42px;width:217px;">Stainless steel dissecting scissors, 10 cm, straight</td>
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			<td height="42" style="height:42px;width:217px;">Stainless steel Iris forceps, 10 cm, curved tip, serrated</td>
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generation of tumor organoids from genetically engineered mouse models of prostate cancer

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) are a rigorous method for studying mammalian development and disease. Our laboratory has developed several GEMMs of prostate cancer (PCa) that lack expression of one or multiple tumor suppressor genes using the site-specific Cre-loxP recombinase system and a prostate-specific promoter. In this article, we describe our method for necropsy of these PCa GEMMs, primarily focusing on dissection of mouse prostate tumors. New methods developed over the last decade have facilitated the culture of epithelial-derived cells to model organ systems in vitro in three dimensions. We also detail a 3D cell culture method to generate tumor organoids from mouse PCa GEMMs. Pre-clinical cancer research has been dominated by 2D cell culture and cell line-derived or patient-derived xenograft models. These methods lack tumor microenvironment, a limitation of using these techniques in pre-clinical studies. GEMMs are more physiologically-relevant for understanding tumorigenesis and cancer progression. Tumor organoid culture is an in vitro model system that recapitulates tumor architecture and cell lineage characteristics. In addition, 3D cell culture methods allow for growth of normal cells for comparison to tumor cell cultures, rarely possible using 2D cell culture techniques. In combination, use of GEMMs and 3D cell culture in pre-clinical studies has the potential to improve our understanding of cancer biology.&#160;

Representative necropsy images of a mouse with a large fluid-filled primary prostate tumor in the anterior prostate region are shown in Figure 2A. In contrast, Figure 2B, shows representative necropsy images of a mouse with a large solid primary prostate tumor for which individual prostate regions are indistinguishable. Fluorescent dissection images show the same solid prostate tumor from Figure 2<s...

Watch this Scientific Journal Video about Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer at JoVE.com

Generation of Tumor Organoids from Genetically Engineered Mouse Models of Prostate Cancer

We show a method for necropsy and dissection of mouse prostate cancer models, focusing on prostate tumor dissection. A step-by-step protocol for generation of mouse prostate tumor organoids is also presented.

Methods based on homologous recombination to modify genes have significantly furthered biological research. Genetically engineered mouse models (GEMMs) ...

Mouse organoid cultures more closely resemble in vivo cell organization than traditional cell cultures. In cancer research, organoids model the original tumor better than 2D cell lines and can be used to test potential cancer therapies in vitro to develop new drug treatments. For en bloc extraction of the male mouse urogenital system grasp the fat pad on either side of the bladder and pull upward to expose the testicle.Carefully dissect each testicle away from the rest of the urogenital region. Gently lift the bladder so that the urogenital region is elevated together, exposing the urethra underneath. While holding the bladder, orient the scissors against the underside of the dorsal prostate to incise the urethra.The entire urogenital region will be released from the abdominal cavity. After removing the urogenital system, the pelvic lymph nodes positioned immediately behind the urogenital system and on either side of the spine will be exposed. To remove the lymph nodes orient straight forceps under the lymph tissue and pull up.Next, grasp the rectum with the straight forceps and cut, pulling up on the rectum to unravel the entire colon and small intestine to look for metastatic lesions in the mesenteric lymph nodes. When the entire ilium has been removed continue to pull on the duodenum to expose the stomach. Cut the esophagus to completely remove the stomach.If any metastatic lesions in the lymph nodes are observed, carefully dissect these nodes away from the intestine for storage in PBS. When the stomach has been removed, the spleen can be harvested from the dorsal side of the abdomen for storage in 4%paraformaldehyde. Remove the liver for storage in PBS.Remove the kidneys from either side of the spine along with any renal lymph nodes containing metastatic lesions. To expose the thoracic cavity carefully cut the diaphragm along the rib cage. The negative pressure released within the thoracic cavity will expose the heart and lungs.Pull up on the sternum to open the thoracic cavity further. Scan the ventral face of the thoracic cavity along the rib cage for metastatic lesions in the thoracic lymph nodes for dissection if present. While still holding the sternum, cut away the ventral rib cage to access the heart and lungs and pull up on the heart to cut underneath the lungs.To fully remove the heart and lungs en bloc cut all of the anterior blood vessels in the trachea. Carefully transfer the heart without damaging the lung tissue or lung metastatic lesions into a container of PBS. For dissection of the prostate tumor place the urogenital region under a dissection microscope.Use a pair of straight forceps and a pair of curved forceps to flip the urogenital region onto its dorsal surface. Locate the proximal prostate region that can be identified by the pink-red color of the urethra. Grasp the urethra firmly to allow manipulation of the urogenital tissue with the curved forceps.Locate the base of the seminal vesicles to allow their careful removal. Then use the curved side of the forceps to remove the vas deferens and as much fatty and connective tissue as possible. While still firmly holding the urethra and proximal prostate region, use a pair of fine pointed scissors to remove the bladder from the urethra.Holding the proximal prostate region and urethra firmly with the straight forceps grasp the anterior prostate region with the curved side of the forceps to firmly pull the tissue away from the bladder and the rest of the prostate. Place the anterior prostate tissue in PBS and locate the ventral prostate region. Remove the ventral prostate region in the same manner.If the lateral region can be distinguished from the dorsal region remove the lateral prostate region on both sides. Then remove the dorsal prostate region and place the proximal prostate region in 4%paraformaldehyde. After mincing, place the tumor pieces into a 15-milliliter tube of digestion buffer for a one-and-a-half to two hour digestion at 37 degrees Celsius.At the end of the digestion, sediment the digested tissue by centrifugation and discard the supernatant. Flick the tube to loosen the cell pellet. Resuspend the cells in one milliliter of pre-warmed trypsin supplemented with 10 micromolar Y-27632 ROCK inhibitor for five minutes at 37 degrees Celsius.At the end of the incubation triturate the tissue slurry five times with a standard P1000 pipette tip. Return the tube to the water bath for an additional five minute incubation and trituration. For matrix dome plating wash the digested cells in nine milliliters of cold medium, then collect the cells by centrifugation.Resuspend the cells in one milliliter of fresh medium for counting. And after counting, collect the cells by centrifugation once again. Dilute the tumor cells in the appropriate volume of matrix.Carefully drop 200 microliters of matrix cell solution into each well of a 55 degree Celsius warmed six-well plate. Allow the domes to solidify at room temperature for two minutes before placing the plate upside down in a 37 degrees Celsius incubator for 20 minutes. When the domes have fully solidified add two milliliters of prostate organoid medium supplemented with androgen R1881 and ROCK inhibitor to each well.Return the plate to the cell culture incubator. Fluorescent dissection images reveal green fluorescent protein, or GFP expression, by solid prostate tumors, indicating that the tumor cells express Cre. The liver and lungs from the same animal exhibit metastatic tumors expressing GFP demonstrating that these tumors originated from the primary prostate tumor.The pelvic lymph node from this mouse expresses GFP and not Tomato, indicating that this metastatic tumor has overtaken the lymph node and no normal tissue remains. At day one of culture small organoids generated from a solid prostate tumor begin to form with both Tomato and GFP-expressing cells present in the tumor organoid culture. By day seven and beyond when the prostate tumor organoids have fully formed however, the organoids express GFP, but not Tomato, suggesting that the organoids have originated from Cre-expressing tumor cells and not from normal epithelial cells.On day one of culture of small organoids generated from fluid-filled prostate tumor both Tomato and GFP-expressing cells are present within the organoid culture. Organoids from fluid-filled prostate tumors express either GFP or Tomato at day seven and beyond however, indicating that the organoids formed from cells that do not express Cre. To stay oriented when harvesting the prostate tissue, be aware of the bladder and urethra positions at all times.After their generation the organoids can be used in imaging applications, molecular biology analysis and drug screens.

generation-tumor-organoids-from-genetically-engineered-mouse-models

Research

JoVE Journal

Genetics

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Simultaneous Video-EEG-ECG Monitoring to Identify Neurocardiac Dysfunction in Mouse Models of Epilepsy

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can potentially increase risk of sudden unexpected death in epilepsy (SUDEP). Electroencephalography (EEG) and electrocardiography (ECG) are widely used clinical diagnostic tools to monitor for abnormal brain and cardiac rhythms in patients. Here, a technique to simultaneously record video, EEG, and ECG in mice to measure behavior, brain, and cardiac activities, respectively, is described. The technique described herein utilizes a tethered (i.e., wired) recording configuration in which the implanted electrode on the head of the mouse is hard-wired to the recording equipment. Compared to wireless telemetry recording systems, the tethered arrangement possesses several technical advantages such as a greater possible number of channels for recording EEG or other biopotentials; lower electrode costs; and greater frequency bandwidth (i.e., sampling rate) of recordings. The basics of this technique can also be easily modified to accommodate recording other biosignals, such as electromyography (EMG) or plethysmography for assessment of muscle and respiratory activity, respectively. In addition to describing how to perform the EEG-ECG recordings, we also detail methods to quantify the resulting data for seizures, EEG spectral power, cardiac function, and heart rate variability, which we demonstrate in an example experiment using a mouse with epilepsy due to Kcna1 gene deletion. Video-EEG-ECG monitoring in mouse models of epilepsy or other neurological disease provides a powerful tool to identify dysfunction at the level of the brain, heart, or brain-heart interactions.

Here, we present a protocol to record brain and heart bio signals in mice using simultaneous video, electroencephalography (EEG), and electrocardiography (ECG). We also describe methods to analyze the resulting EEG-ECG recordings for seizures, EEG spectral power, cardiac function, and heart rate variability.

In epilepsy, seizures can evoke cardiac rhythm disturbances such as heart rate changes, conduction blocks, asystoles, and arrhythmias, which can ...

Identification of Homologous Recombination Events in Mouse Embryonic Stem Cells Using Southern Blotting and Polymerase Chain Reaction

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome editing, embryonic stem (ES) cell-based gene-targeting technology does not have these shortcomings and is widely used to modify animal/mouse genome ranging from large deletions/insertions to single nucleotide substitutions. Notably, identifying the relatively few homologous recombination (HR) events necessary to obtain desired ES clones is a key step, which demands accurate and reliable methods. Southern blotting and/or conventional PCR are often utilized for this purpose. Here, we describe the detailed procedures of using those two methods to identify HR events that occurred in mouse ES cells in which the endogenous Myh9 gene is intended to be disrupted and replaced by cDNAs encoding other nonmuscle myosin heavy chain IIs (NMHC IIs). The whole procedure of Southern blotting includes the construction of targeting vector(s), electroporation, drug selection, the expansion and storage of ES cells/clones, the preparation, digestion, and blotting of genomic DNA (gDNA), the hybridization and washing of probe(s), and a final step of autoradiography on the X-ray films. PCR can be performed directly with prepared and diluted gDNA. To obtain ideal results, the probes and restriction enzyme (RE) cutting sites for Southern blotting and the primers for PCR should be carefully planned. Though the execution of Southern blotting is time-consuming and labor-intensive and PCR results have false positives, the correct identification by Southern blotting and the rapid screening by PCR allow the sole or combined application of these methods described in this paper to be widely used and consulted by most labs in the identification of genotypes of ES cells and genetically modified animals.

Here, we present a detailed protocol for identifying homologous recombination events that occurred in mouse embryonic stem cells using Southern blotting and/or PCR. This method is exemplified by the generation of nonmuscle myosin II genetic replacement mouse models using traditional embryonic stem cell-based homologous recombination-mediated targeting technology.

Relative to the issues of off-target effects and the difficulty of inserting a long DNA fragment in the application of designer nucleases for genome ...

Generation of Cancer Cell Clones to Visualize Telomeric Repeat-containing RNA TERRA Expressed from a Single Telomere in Living Cells

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in telomere biology, including heterochromatin formation and telomere length homeostasis. Recent findings revealed that TERRA molecules also interact with internal chromosomal regions to regulate gene expression in mouse embryonic stem (ES) cells. In line with this evidence, RNA fluorescence in situ hybridization (RNA-FISH) analyses have shown that only a subset of TERRA transcripts localize at chromosome ends. A better understanding of the dynamics of TERRA molecules will help define their function and mechanisms of action. Here, we describe a method to label and visualize single-telomere TERRA transcripts in cancer cells using the MS2-GFP system. To this aim, we present a protocol to generate stable clones, using the AGS human stomach cancer cell line, containing MS2 sequences integrated at a single subtelomere. Transcription of TERRA from the MS2-tagged telomere results in the expression of MS2-tagged TERRA molecules that are visualized by live-cell fluorescence microscopy upon co-expression of a MS2 RNA-binding protein fused to GFP (MS2-GFP). This approach enables researchers to study the dynamics of single-telomere TERRA molecules in cancer cells, and it can be applied to other cell lines.

Here, we present a protocol to generate cancer cell clones containing a MS2 sequence tag at a single subtelomere. This approach, relying on the MS2-GFP system, enables visualization of the endogenous transcripts of telomeric repeat-containing RNA (TERRA) expressed from a single telomere in living cells.

Telomeres are transcribed, giving rise to telomeric repeat-containing long noncoding RNAs (TERRA), which have been proposed to play important roles in ...

Intraventricular Transplantation of Engineered Neuronal Precursors for In Vivo Neuroarchitecture Studies

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in vivo gene control of neocortical projection neuron morphology. This method is based on (1) in vitro lentiviral engineering of neuronal precursors as "test" and "control" cells, (2) their co-transplantation into wild-type brains, and (3) paired morphometric evaluation of their neuronal derivatives. Specifically, E12.5 pallial precursors from panneuronal, genetically labeled donors, are employed for this purpose. They are engineered to take advantage of selected promoters and tetON/OFF technology, and they are free-hand transplanted into neonatal lateral ventricles. Later, upon immunofluorescence profiling of recipient brains, silhouettes of transplanted neurons are fed into NeurphologyJ open source software, their morphometric parameters are extracted, and average length and branching index are calculated. Compared to other methods, this one offers three main advantages: it permits achieving of fine control of transgene expression at affordable costs, it only requires basic surgical skills, and it provides statistically reliable results upon analysis of a limited number of animals. Because of its design, however, it is not adequate to address non cell-autonomous control of neuroarchitecture. Moreover, it should be preferably used to investigate neurite morphology control after completion of neuronal migration. In its present formulation, this method is exquisitely tuned to investigate gene control of glutamatergic neocortical neuron architecture. Taking advantage of transgenic lines expressing EGFP in other specific neural cell types, it can be re-purposed to address gene control of their architecture.

Based on in vitro lentiviral engineering of neuronal precursors, their co-transplantation into wild-type brains and paired morphometric evaluation of "test" and "control" derivatives, this method allows accurate modeling of in vivo gene control of neocortical neuron morphology in a simple and affordable way.

Gene control of neuronal cytoarchitecture is currently the subject of intensive investigation. Described here is a simple method developed to study in ...

CRISPR/Cas9 Ribonucleoprotein-mediated Precise Gene Editing by Tube Electroporation

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of CRISPR/Cas9 elements into the target cells by transfection is a prerequisite for efficient gene editing. This protocol demonstrates that tube electroporation (TE) machine-mediated delivery of CRISPR/Cas9 ribonucleoprotein (RNP), along with single-stranded oligodeoxynucleotide (ssODN) donor templates to different types of mammalian cells, leads to robust precise gene editing events. First, TE was applied to deliver CRISPR/Cas9 RNP and ssODNs to induce disease-causing mutations in the interleukin 2 receptor subunit gamma (IL2RG) gene and sepiapterin reductase (SPR) gene in rabbit fibroblast cells. Precise mutation rates of 3.57%-20% were achieved as determined by bacterial TA cloning sequencing. The same strategy was then used in human iPSCs on several clinically relevant genes including epidermal growth factor receptor (EGFR), myosin binding protein C, cardiac (Mybpc3), and hemoglobin subunit beta (HBB). Consistently, highly precise mutation rates were achieved (11.65%-37.92%) as determined by deep sequencing (DeepSeq). The present work demonstrates that tube electroporation of CRISPR/Cas9 RNP represents an efficient transfection protocol for gene editing in mammalian cells.

Presented here is a protocol for efficient CRISPR/Cas9 ribonucleoprotein-mediated gene editing in mammalian cells using tube electroporation.

Gene editing nucleases, represented by CRISPR-associated protein 9 (Cas9), are becoming mainstream tools in biomedical research. Successful delivery of ...

The Use of Mouse Mammary Tumor Cells in an In Vitro Invasion Assay as a Measure of Oncogenic Cell Behavior

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a porous membrane in a process analogous to the initial steps of cancer cell metastasis. The tested cells can be altered for the gene expression or treated with inhibitors to test for changes in the invasion potential. This experiment tests the aggressive phenotype of the mouse mammary tumor cells to discover and characterize the potential oncogenes that promote cell invasion. This technique, however, can be versatile and adapted to many different applications. The experiment itself can be done in one day and the results are acquired by light microscopy in less than a day. The results include counts of the number of invading cells for comparison and analysis. The in vitro invasion assay is a rapid, inexpensive, and clear-cut method for determining cell behavior in a culture that can be used as an initial assessment before more involved in vivo assays.

The in vitro cell invasion assay is used to measure the potential of cancer metastasis by quantifying the cellular potential for invasion and migration using cell culture inserts containing protein matrix. Cells are challenged to migrate through the protein matrix and a porous membrane, towards a chemoattractant, and then quantified by light microscopy.

The in vitro invasion assay uses a protein-rich matrix in a Boyden chamber to measure the ability of cultured cells to pass through the matrix and a ...

RNA Next-Generation Sequencing and a Bioinformatics Pipeline to Identify Expressed LINE-1s at the Locus-Specific Level

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and mutagenesis. Understanding the expression patterns of L1 loci at the individual level will lend to the understanding of the biology of this mutagenic element. This autonomous element makes up a significant portion of the human genome with over 500,000 copies, though 99% are truncated and defective. However, their abundance and dominant number of defective copies make it challenging to identify authentically expressed L1s from L1-related sequences expressed as part of other genes. It is also challenging to identify which specific L1 locus is expressed due to the repetitive nature of the elements. Overcoming these challenges, we present an RNA-Seq bioinformatic approach to identify L1 expression at the locus specific level. In summary, we collect cytoplasmic RNA, select for polyadenylated transcripts, and utilize strand-specific RNA-Seq analyses to uniquely map reads to L1 loci in the human reference genome. We visually curate each L1 locus with uniquely mapped reads to confirm transcription from its own promoter and adjust mapped transcript reads to account for mappability of each individual L1 locus. This approach was applied to a prostate tumor cell line, DU145, to demonstrate the ability of this protocol to detect expression from a small number of the full-length L1 elements.

Here, we present a bioinformatic approach and analyses to identify LINE-1 expression at the locus specific level.

Long INterspersed Elements-1 (LINEs/L1s) are repetitive elements that can copy and randomly insert in the genome resulting in genomic instability and ...

Tumorsphere Derivation and Treatment from Primary Tumor Cells Isolated from Mouse Rhabdomyosarcomas

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children. Although significant efforts have enabled the identification of common mutations associated with RMS and allowed discrimination of different RMS subtypes, major challenges still exist for the development of novel treatments to further improve prognosis. Although identified by the expression of myogenic markers, there is still significant controversy over whether RMS has myogenic or non-myogenic origins, as the cell of origin is still poorly understood. In the present study, a reliable method is provided for the tumorsphere assay for mouse RMS. The assay is based on functional properties of tumor cells and allows the identification of rare populations in the tumor with tumorigenic functions. Also described are procedures for testing recombinant proteins, integrating transfection protocols with the tumorsphere assay, and evaluating candidate genes involved in tumor development and growth. Described further is a procedure for allograft transplantation of tumorspheres into recipient mice to validate tumorigenic function in vivo. Overall, the described method allows reliable identification and testing of rare RMS tumorigenic populations that can be applied to RMS arising in different contexts. Finally, the protocol can be utilized as a platform for drug screening and future development of therapeutics.

This protocol describes a reproducible method for isolation of mouse rhabdomyosarcoma primary cells, tumorsphere formation and treatment, and allograft transplantation starting from tumorspheres cultures.