Name: Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene (Video) | JoVE Journal
Uploaded: 2014-12-31T15:00:00.000+00:00
Duration: 8 min 18 s
Description: Learn the focus formation assay to evaluate oncogenic potential using retroviral vectors and NIH 3T3 mouse fibroblast cells. This method enables researchers to screen candidate oncogenes for transforming activity.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This work was supported by a grant from the NIH Director&#39;s New Innovator Award Program (E.D.). A. A. was supported in part by undergraduate awards from the National Cancer Institute and the National Science Foundation.

1. Making the Viral Vectors
The coding sequences for the gene of interest, as well as the positive and negative controls, are inserted into pBABEpuro by traditional cloning methods (PCR amplification, restriction digestion and ligation). There are four restriction sites on the vector where the DNA can be inserted: BamHI, SnaBI, EcoRI and SalI.
<ol>
	<li>Prepare transfection-grade plasmid DNA from competent cells using the QIAGEN plasmid midi kit.</li>
	<li>Measure DNA concentration using a NanoDrop2000 spectrophotometer.</li>
</ol>
2. Retrovirus Production
The Plat-E packaging cell line will be used to produce ecotropic retrovirus that will deliver the cDNA of interest to NIH 3T3 cells.
<ol>
	<li>Establishing Plat-E Cultures from Frozen Cells
	<ol>
		<li>Quickly thaw Plat-E cells in a 37 &#176;C water bath and transfer to a 15 ml conical tube. Slowly add 9 ml of Plat-E medium (Dulbecco&#8217;s Modified Eagle Medium (DMEM) + 10% Fetal Bovine Serum (FBS) + Penicillin and Streptomycin).</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Re-suspend the cells with 10 ml of Plat-E medium and transfer to a 10 cm culture dish.</li>
		<li>Incubate the cells in a 37 &#176;C, 5% CO2 incubator until they are 80-90% confluent (about 2 days).</li>
	</ol>
	</li>
	<li>Cell Splitting
	<ol>
		<li>Aspirate the medium and wash cells once with PBS.</li>
		<li>Add 2 ml of 0.05% trypsin-EDTA and incubate for 1 min at RT.</li>
		<li>Detach the cells by finger tapping, add 10 ml of Plat-E medium, and transfer the cell suspension to a 15 ml tube.</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cells with 10 ml of Plat-E medium and seed them in new dishes at 1:4-1:6 dilutions.</li>
	</ol>
	</li>
	<li>Plat-E Seeding and Transfection
	<ol>
		<li>Seed 2 x 106 cells per 10 cm culture dish using Plat-E medium without any antibiotics.</li>
		<li>Incubate the cells O/N in a 37 &#176;C, 5% CO2 incubator.</li>
		<li>Next day, transfer 300 &#956;l of Opti-MEM into 1.5 ml microfuge tube.</li>
		<li>Add 27 &#956;l of polyethylenimine (PEI), a cost-effective transfection reagent12, to the prepared tube with Opti-MEM. Mix gently by finger tapping and incubate for 5 min at RT.</li>
		<li>Add 9 &#956;g of transfection-grade plasmid DNA into the Opti-MEM/PEI tube, mix gently by vortexing, and incubate for 15 min at RT.</li>
		<li>Add the DNA/PEI complex drop-wise into the Plat-E dish, and incubate O/N&#160;at 37 &#176;C, 5% CO2.</li>
		<li>Next day, aspirate the medium containing the transfection reagent and add 10 ml of fresh Plat-E medium (without antibiotics).</li>
		<li>Return the cells to the incubator.</li>
	</ol>
	</li>
</ol>
3. NIH 3T3 Cells and Infection
<ol>
	<li>Establishing NIH 3T3 Cultures and Seeding
	<ol>
		<li>Quickly thaw NIH 3T3 cells in a 37 &#176;C water bath and transfer to a 15 ml tube. Slowly add 9 ml of NIH 3T3 medium (DMEM + 10% FBS).</li>
		<li>Centrifuge the tube at 180 x g for 5 min and discard the supernatant.</li>
		<li>Resuspend the cells with 10 ml of NIH 3T3 medium and transfer to a 10 cm culture dish.</li>
		<li>Incubate the cells in a 37 &#176;C, 5% CO2 incubator. It is very important that NIH 3T3 cells are always kept underconfluent (50-60%), as the frequency of spontaneous transformation (and therefore basal levels of foci formation) increases once a culture reaches confluency.</li>
		<li>Seed 3 x 105 cells per 10 cm dish and incubate O/N for infection the next day.</li>
	</ol>
	</li>
	<li>Infection
	<ol>
		<li>Harvest the retroviral supernatant from the Plat-E dish by using a 10 ml disposable syringe, filtering it through a 0.45 &#956;m pore size nylon membrane filter, and transferring it into a 15 ml tube.</li>
		<li>Add 10 ml of fresh Plat-E medium to the cells (without antibiotics), and return them to the incubator.</li>
		<li>Aspirate the medium from the NIH 3T3 cell dish to be infected, and add 5 ml of regular NIH 3T3 medium and 5 ml of virus-containing filtered supernatant.</li>
		<li>Add polybrene to the dish at a concentration of 6 &#956;g/ml.</li>
		<li>Incubate the cells O/N at 37 &#176;C, 5% CO2.</li>
		<li>Next day, repeat steps 3.2.1 &#8211; 3.2.5 for a second round of infection.</li>
		<li>Replace the virus-containing medium with regular NIH 3T3 medium.</li>
		<li>Allow the NIH 3T3 cells to grow for 2-3 weeks, replacing the medium as necessary. Check expression of the protein of interest by conventional protein electrophoresis and immunoblot on whole cell lysates from replica plates.</li>
	</ol>
	</li>
</ol>
4. Staining and Quantification
<ol>
	<li>Crystal Violet Staining
	<ol>
		<li>Aspirate the NIH 3T3 medium and place the dishes on ice.</li>
		<li>Wash the dishes twice with ice-cold PBS.</li>
		<li>Fix the cells with ice-cold methanol for 10 min.</li>
		<li>Remove the dishes from ice, aspirate the methanol, and add 3 ml of 0.5% crystal violet solution made in 25% methanol (RT).</li>
		<li>Incubate the dishes for 5 min at RT.</li>
		<li>Aspirate the crystal violet solution and carefully rinse the dish with Milli-Q H2O until no color comes off in the rinse.</li>
		<li>Allow the dishes to dry O/N&#160;on a benchtop (uncovered).</li>
	</ol>
	</li>
	<li>Foci Quantification
	<ol>
		<li>Once the dishes are dry, use a ruler to measure the darkly colored collections of cells on the plate. Only count those that are greater than 5 mm in diameter as &#8220;foci.&#8221; Record the number of foci and calculate averages and the significance compared to the controls.</li>
	</ol>
	</li>
</ol>

<ol><li>Clark, G. J., Cox, A. D., Graham, S. M., Der, C. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Biological+assays+for+Ras+transformation%5BTitle%5D)+AND+%22Methods+in+enzymology%22%5BJournal%5D)">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li>
<li>Celis, J. E. <a target="_blank" href="http://scholar.google.com/scholar?hl=en&safe=off&q=author%3aCelis%2C+JE+%22Cell+biology+%3A+a+laboratory+handbook%22">Cell biology : a laboratory handbook</a>. , 3rd edn, Elsevier Academic. 345-352 (2006).</li>
<li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Neoplastic+transformation+assays%5BTitle%5D)+AND+%22Methods+in+molecular+biology%22%5BJournal%5D)">Neoplastic transformation assays.</a> Methods in molecular biology. 165, 151-164 (2001).</li>
<li>Johnson, P. J., Coussens, P. M., Danko, A. V., Shalloway, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells%5BTitle%5D)+AND+%22Molecular+and+cellular+biology%22%5BJournal%5D)">Overexpressed pp60c-src can induce focus formation without complete transformation of NIH 3T3 cells.</a> Molecular and cellular biology. 5, 1073-1083 (1985).</li>
<li>Bonner, T. I., Kerby, S. B., Sutrave, P., Gunnell, M. A., Mark, G., Rapp, U. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Structure+and+biological+activity+of+human+homologs+of+the+raf%2Fmil+oncogene%5BTitle%5D)+AND+%22IMolecular+and+cellular+biology%22%5BJournal%5D)">Structure and biological activity of human homologs of the raf/mil oncogene.</a> IMolecular and cellular biology. 5, 71400-71407 (1985).</li>
<li>Yancopoulos, G. D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture%5BTitle%5D)+AND+%22Proceedings+of+the+National+Academy+of+Sciences+of+the+United+States+of+America%22%5BJournal%5D)">N-myc can cooperate with ras to transform normal cells in culture.</a> Proceedings of the National Academy of Sciences of the United States of America. 82, 5455-5459 (1985).</li>
<li>Fujiwara, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Transforming+activity+of+purinergic+receptor+P2Y%2C+G+protein+coupled%2C+8+revealed+by+retroviral+expression+screening%5BTitle%5D)+AND+%22Leukemia+%26+lymphoma%22%5BJournal%5D)">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia & lymphoma. 48, 978-986 (2007).</li>
<li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plat-E%3A+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses%5BTitle%5D)+AND+%22Gene%22%5BJournal%5D)">Plat-E: an efficient and stable system for transient packaging of retroviruses.</a> Gene. 7, 1063-1066 (2000).</li>
<li>Morgenstern, J. P., Land, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Advanced+mammalian+gene+transfer%3A+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line%5BTitle%5D)+AND+%22Nucleic+acids+research%22%5BJournal%5D)">Advanced mammalian gene transfer: high titre retroviral vectors with multiple drug selection markers and a complementary helper-free packaging cell line.</a> Nucleic acids research. 18, 3587-3596 (1990).</li>
<li>Kim, T. K., Eberwine, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mammalian+cell+transfection%3A+the+present+and+the+future%5BTitle%5D)+AND+%22Analytical+and+bioanalytical+chemistry%22%5BJournal%5D)">Mammalian cell transfection: the present and the future.</a> Analytical and bioanalytical chemistry. 397, 3173-3178 (2010).</li>
<li>Klaver, B., Berkhout, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Comparison+of+5%27+and+3%27+long+terminal+repeat+promoter+function+in+human+immunodeficiency+virus%5BTitle%5D)+AND+%22Journal+of+virology%22%5BJournal%5D)">Comparison of 5' and 3' long terminal repeat promoter function in human immunodeficiency virus.</a> Journal of virology. 68, 3830-3840 (1994).</li>
<li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Cost-effective+gene+transfection+by+DNA+compaction+at+pH+4.0+using+acidified%2C+long+shelf-life+polyethylenimine%5BTitle%5D)+AND+%22Cytotechnology%22%5BJournal%5D)">Cost-effective gene transfection by DNA compaction at pH 4.0 using acidified, long shelf-life polyethylenimine.</a> Cytotechnology. 62, 73-82 (2010).</li>
<li>Fox, E. J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells%5BTitle%5D)+AND+%22The+Biochemical+journal%22%5BJournal%5D)">S-phase-specific expression of the Mad3 gene in proliferating and differentiating cells.</a> The Biochemical journal. 359, 361-367 (2001).</li>
<li>Yun, J. S., Rust, J. M., Ishimaru, T., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+novel+role+of+the+Mad+family+member+Mad3+in+cerebellar+granule+neuron+precursor+proliferation%5BTitle%5D)+AND+%22Molecular+and+cellular+biology%22%5BJournal%5D)">A novel role of the Mad family member Mad3 in cerebellar granule neuron precursor proliferation.</a> Molecular and cellular biology. 27, 8178-8189 (2007).</li>
<li>Gore, Y., Lantner, F., Hart, G., Shachar, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mad3+negatively+regulates+B+cell+differentiation+in+the+spleen+by+inducing+Id2+expression%5BTitle%5D)+AND+%22Molecular+biology+of+the+cell%22%5BJournal%5D)">Mad3 negatively regulates B cell differentiation in the spleen by inducing Id2 expression.</a> Molecular biology of the cell. 21, 1864-1871 (2010).</li>
<li>Barisone, G. A., Yun, J. S., Diaz, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((From+cerebellar+proliferation+to+tumorigenesis%3A+new+insights+into+the+role+of+Mad3%5BTitle%5D)+AND+%22Cell+cycle%22%5BJournal%5D)">From cerebellar proliferation to tumorigenesis: new insights into the role of Mad3.</a> Cell cycle. 7, 423-427 (2008).</li>
<li>Barisone, G. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Role+of+MXD3+in+proliferation+of+DAOY+human+medulloblastoma+cells%5BTitle%5D)+AND+%22PloS+One%22%5BJournal%5D)">Role of MXD3 in proliferation of DAOY human medulloblastoma cells.</a> PloS One. 7, e38508(2012).</li>
<li>Nair, S. K., Burley, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((X-ray+structures+of+Myc-Max+and+Mad-Max+recognizing+DNA.+Molecular+bases+of+regulation+by+proto-oncogenic+transcription+factors%5BTitle%5D)+AND+%22Cell%22%5BJournal%5D)">X-ray structures of Myc-Max and Mad-Max recognizing DNA. Molecular bases of regulation by proto-oncogenic transcription factors.</a> Cell. 112, 193-205 (2003).</li>
</ol>

The authors have nothing to disclose.

The FFA provides a quick and easy method to evaluate malignant transformation in vitro. It is amenable to screening of a relatively large number of candidate genes, and its modest technical requirements make it cost-effective. Furthermore, two or more genes can be coexpressed (sometimes referred to as a &#8220;cooperation&#8221; assay) to evaluate the tumorigenic potential of the combination. The advantages of this assay rely on its straightforward technique, its ease of quantification and its relatively short t...

Tumor cells can be distinguished from their normal counterparts by a wide range of alterations, from patterns of gene expression to epigenomics to morphological and proliferative changes. Among the latter, reduced dependence on serum, loss of contact (density) inhibition, the acquisition of anchorage-independent proliferation and, ultimately, the ability to form tumors when injected into animals are useful, measurable indicators of malignant transformation3. Several in vitro and in vivo assays have been developed for cellular transformation. In vitro assays aim at identifying and measuring changes in culture morphology (focus formation assay), culture dynamics (growth rate, saturation density) and growth factor (growth in reduced serum) or anchorage (growth in soft agar) requirements. The gold standard for determining the malignant nature of a cell type remains tumor formation (xenografts) in experimental animals. However, the cost and length of in vivo studies do not always make them justifiable as a first validation step or screening of candidate oncogenes. Although no in vitro assay provides a definite assessment of the oncogenic potential of a gene, they do provide insight into oncogenic potential that may narrow down future in vivo studies. One of the most widely used systems for evaluating oncogenic potential in vitro is the Focus Formation Assay2. This approach relies on the use of NIH 3T3 mouse fibroblast, a non-transformed cell line that shows strong contact inhibition. Overexpression of an oncogene results in loss of density-dependent growth; transformed cells can then grow in multiple layers, forming &#8220;foci&#8221;, easily visualized against the background monolayer of non-transformed cells. The Focus Formation Assay, then, measures the ability of a candidate oncogene to induce malignant transformation, as evidenced by the loss of contact inhibition as a measurable phenotype. The FFA has been used to evaluate transformation by overexpression of protein kinases (e.g., Src4, BRAF5), transcription factors (e.g., N-myc6), G-protein-coupled receptors (e.g., P2RY87) and GTPases (e.g., Ras1), among others. The relative ease of this assay makes it a good choice that will provide a quick and visually clear answer to whether overexpression of the gene is sufficient to transform NIH 3T3 mouse fibroblast cells in vitro.
The FFA described in this protocol uses the Plat-E packaging cell line8, which provides viral packaging proteins, and the retroviral vector pBABEpuro9 (Addgene plasmid 1764) to produce retrovirus. After transfection with the pBABEpuro construct containing the gene of interest, the Plat-E cell line will produce ecotropic retrovirus that can be used to infect NIH 3T3 cells. This viral method of gene delivery is more efficient than traditional chemical transfection methods and it offers a way to sustainably express the gene10. Once incorporated into the genome of the NIH 3T3 cells, overexpression of the gene of interest is driven by the viral long terminal repeats (LTR) promoter11. This constant expression can be used to determine whether the gene of interest has oncogenic activity, as measured by the formation of foci, on the NIH 3T3 cells.

<table><tbody><tr><td>pBABE-puro vector</td><td>Addgene</td><td>Plasmid 1764</td><td>cloning vector</td></tr><tr><td>Platinum-E Retroviral Packaging Cell Line, Ecotropic</td><td>Cell Biolabs, Inc.</td><td>RV-101</td><td>cell line for viral production</td></tr><tr><td>NIH 3T3 Cell Line murine</td><td>Sigma-Aldrich</td><td>93061524</td><td>cell line for focus formation assay</td></tr><tr><td>10 ml BD Luer-Lok tip syringe</td><td>BD Biosciences</td><td>309604</td><td>viral production reagent</td></tr><tr><td>0.45 &amp;mu;m Puradisc Syringe Filter</td><td>Whatman</td><td>6750-2504</td><td>viral production reagent</td></tr><tr><td>Polyethylenimine (PEI)</td><td>Polysciences, Inc.</td><td>23966-2</td><td>cell transfection reagent</td></tr><tr><td>Polybrene Infection / Transfection Reagent</td><td>EMD Millipore</td><td>TR-1003-G</td><td>cell transfection reagent</td></tr><tr><td>Crystal Violet</td><td>Fisher Scientific</td><td>C581-25</td><td>cell stain reagent</td></tr><tr><td>Plasmid Plus Midi Kit</td><td>QIAGEN</td><td>12945</td><td>plasmid purification</td></tr><tr><td>BD Falcon Tissue Culture Dishes</td><td>BD Biosciences</td><td>353003</td><td>cell culture supplies</td></tr><tr><td>Dulbecco&#039;s Modified Eagle Medium (DMEM)</td><td>Gibco</td><td>11995-065</td><td>cell culture media</td></tr><tr><td>0.05% Trypsin-EDTA</td><td>Gibco</td><td>25300-054</td><td>cell culture supplies</td></tr><tr><td>Opti-MEM I Reduced Serum Medium</td><td>Gibco</td><td>31985-062</td><td>cell culture media</td></tr><tr><td>Fetal Bovine Serum (FBS)</td><td>Gibco</td><td>16000-044</td><td>cell culture media</td></tr></tbody></table>

focus formation: a cell-based assay to determine the oncogenic potential of a gene

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent growth potential, and the ability to form tumors in experimental animals1. In NIH 3T3 cells, the Ras signal transduction pathway is known to trigger many of these events, what is known as Ras transformation. The introduction of an overexpressed gene in NIH 3T3 cells may promote morphological transformation and loss of contact inhibition, which can help determine the oncogenic potential of that gene of interest. An assay that provides a straightforward method to assess one aspect of the transforming potential of an oncogene is the Focus Formation Assay (FFA)2. When NIH 3T3 cells divide normally in culture, they do so until they reach a confluent monolayer. However, in the presence of an overexpressed oncogene, these cells can begin to grow in dense, multilayered foci1 that can be visualized and quantified by crystal violet or Hema 3 staining. In this article we describe the FFA protocol with retroviral transduction of the gene of interest into NIH 3T3 cells, and how to quantify the number of foci through staining. Retroviral transduction offers a more efficient method of gene delivery than transfection, and the use of an ecotropic murine retrovirus provides a biosafety control when working with potential human oncogenes.

MXD3 is a basic helix-loop-helix leucine zipper (bHLHZ) transcription factor that is a member of the MYC/MAX/MAD network. It is an atypical member of the MAD family13-15, and it has been reported to be involved in carcinogenesis16,17. When compared to pBABEpuro (negative control) and MYC (positive control), the NIH 3T3 dishes where MXD3 was overexpressed had significantly less foci (Figure 1A). The data in Figure 1B were collected from multiple experiments to determ...

The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.

Malignant transformation of cells is typically associated with increased proliferation, loss of contact inhibition, acquisition of anchorage-independent ...

The overall goal of this procedure is to perform the focus formation assay in mouse fibroblast cell lines, using a retrovirus to determine the oncogenic potential of a gene. This is accomplished by first cloning the sequence for the gene of interest into a retroviral vector. The second step is to produce retroviral particles using the viral vector and PLA e packaging cells.Next, the retrovirus is used to infect NIH three T three mouse fibroblast cells in order to assess the transforming potential of the gene of interest. The final step is to allow the NIH three T three cells to divide for two to three weeks, followed by crystal violet staining to quantify the number of foci formed. Ultimately, the focus formation assay is used to show if overexpression of a gene of interest is sufficient to induce density independent growth in mouse fibroblast cells.This method can help answer key questions in the field of cancer biology, such as screening for the oncogenic potential of a gene. To begin, make the viral vector as described in the text protocol. Then establish plat E cultures from frozen cells by quickly thawing plat E cells in a 37 degree Celsius water bath, and then transferring to a 15 milliliter conical tube.Slowly add nine milliliters of PLA E medium to the 15 milliliter conical tube. Centrifuge the tube at 180 times G for five minutes, and discard the supernatant. Resuspend the cells with 10 milliliters of plat E medium, and transfer to a 10 centimeter culture dish.Then incubate the cells in a 37 degree Celsius 5%CO2 incubator until they are between 80 and 90%Confluent. Perform cells splitting by first aspirating the medium, then wash the cells once with PBS before adding two milliliters of 0.05%tripsin EDTA and incubating for one minute at room temperature, detach the cells by finger tapping. Then add 10 milliliters of plat E medium and transfer the cell suspension to a 15 milliliter tube.Centrifuge the tube as before and resuspend the cells with 10 milliliters of plat E medium. Next seed, 2 million cells per 10 centimeter culture dish using plat E medium without any antibiotics and leave cells to incubate overnight. The next day, transfer 300 microliters of optimum into a 1.5 milliliter micro tube.Add 27 microliters of polyethylene amine to the prepared tube with Optum. Mix gently by finger tapping and incubate for five minutes at room temperature. Next, add nine micrograms of transfection grade plasmid DNA into the tube mixed gently by vortexing and incubate for 15 minutes at room temperature.Then add the mixture dropwise into the plat E dish before incubating overnight. The next day aspirate the medium containing the transfection reagent and add 10 milliliters of fresh plat E medium before returning the cells to the incubator to establish NIH three T three cultures. First, quickly thaw the cells in a 37 degree Celsius water bath and transfer to a 15 milliliter tube.Slowly add nine milliliters of NIH three, T three medium to the cells, centrifuge the tube at 180 times G for five minutes and discard the snat. Then resuspend the cells with 10 milliliters of NIH three, T three medium, and transfer to a 10 centimeter culture dish. Incubate the cells in a 37 degree Celsius 5%CO2 incubator, keeping them under confluent as the frequency of spontaneous transformation increases.Once a culture reaches co fluency, once grown seed three times 10 to the fifth nih, three T three cells per 10 centimeter dish and incubate overnight for infection the next day. The next day, harvest the retroviral supernatant from the plat E dish by using a 10 milliliter disposable syringe, filtering it through a 0.45 micron pore size nylon membrane filter, and transferring it into a 15 milliliter tube. Add 10 milliliters of fresh plat E medium to the cells before returning them to the incubator.Then aspirate the medium from the NIH three T three cell dish to be infected and add five milliliters of regular NI H three, T three medium, and five milliliters of virus containing filtered supernatant. Next, add poly brain to the dish. Add a concentration of six micrograms per milliliter.Incubate the cells overnight before repeating the infection steps for a second round of infection. Following infection, replace the virus, contain medium with regular IH three T three medium. Allow the IH three T three cells to grow for two to three, replacing the medium as necessary for crystal violet staining.Aspirate the NIH three T three medium and place the dishes on ice. Wash the dishes twice with ice cold PBS. Fix the cells with ice cold methanol for 10 minutes.Then remove the dishes from ice and aspirate the methanol. Add three milliliters of 0.5%crystal violet solution made in 25%methanol and incubate the dishes for five minutes at room temperature. Following incubation, aspirate the crystal violet solution and carefully rinse the dish with Milli Q water until no color comes off in the rinse.Allow the dishes to dry uncovered overnight on a bench top. Once the dishes are dry, use a ruler to measure the foci and count those that are greater than five millimeters in diameter record. The number MXD three is a basic helix loop helix leucine zipper transcription factor that is an atypical member of the MAD family, and it has been reported to be involved in carcinogenesis when compared to the negative control and the positive control.The NIH three T three dishes where MXD three was overexpressed had significantly less foci. The data were collected from multiple experiments to determine significance and the same trends were observed from this assay. Results suggest that MXD three does not function as an oncogene Following this procedure.Other methods like focus formation assay with gene combinations, growth in soft auger proliferation and cell cycle analysis can be performed in order to answer additional questions To assess the role of a gene of interest in oncogenesis pathogenesis.

focus-formation-cell-based-assay-to-determine-oncogenic-potential

Research

JoVE Journal

Medicine

This video describes a soft agar colony formation assay to test the effects of a PADI enzyme inhibitor and BB-Cl-amidine on breast cancer tumorigenicity in vitro. This assay also enables the quantitative assessment of cell transformation potential within the context of genetic perturbations.

Soft Agar Colony Formation Assay: A Method to Test Effects of Novel Compounds on Cancer Cell Proliferation

This video describes the colony formation assay, which helps determine lung cancer cells' ability to proliferate indefinitely and form individual colonies. We can analyze the effect of an anticancer agent on the colony-forming cells by analyzing the changes in the number and/or size of the resulting colonies.

Colony Formation Assay: Assessing the Efficacy of Anticancer Agents on Colony-Forming Lung Cancer Cells

This video describes the protocol for methyl cellulose-based colony formation unit or CFU assay, which helps identify and count the number of colonies formed by leukemic cells in response to the treatment with anticancer compounds.

Methyl Cellulose-Based CFU Assay: A Method to Determine the Effect of Anti-cancer Compounds on Colony Formation in Leukemic Cells

Achieving a systems level understanding of cellular processes is a goal of modern-day cell biology. We describe here strategies for multiplexing luciferase reporters of various cellular function end-points to interrogate gene function using genome-scale RNAi libraries.

A Multiplexed Luciferase-based Screening Platform for Interrogating Cancer-associated Signal Transduction in Cultured Cells

The soft agar colony formation assay is a method used to confirm cellular anchorage-independent growth in vitro. The goal of this protocol is to illustrate a stringent method for the detection of the tumorigenic potential of transformed cells and the tumor suppressive effects of proteins on transformed cells.

The Soft Agar Colony Formation Assay

Identifying novel drug targets that transition from pre-clinical testing to human trials is a scientific priority. To that end, here we describe a functional genomics approach for examining the impact of gene depletion on cancer cell line spheroids, which more appropriately model human cancers in vivo.

Utilizing Functional Genomics Screening to Identify Potentially Novel Drug Targets in Cancer Cell Spheroid Cultures

We have developed a novel protocol for studying heterocellular population dynamics in response to perturbations. This manuscript describes an imaging-based platform that produces quantitative datasets for simultaneous characterization of multiple cellular phenotypes of heterocellular populations in a robust manner.

Discrimination and Characterization of Heterocellular Populations Using Quantitative Imaging Techniques

Here, we describe protocols for the genetic and chemical validation of c-Fos and Dusp1 as a drug target in leukemia using in vitro and in vivo genetic and humanized mouse models. This method can be applied to any target for genetic validation and therapeutic development.

Methods for Evaluating the Role of c-Fos and Dusp1 in Oncogene Dependence

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

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

Intrinsically disordered domains are important for oncogenic fusion transcription factor function. To therapeutically target these proteins, a more detailed understanding of the regulatory mechanisms employed by these domains is required. Here, we use transcriptomics to map important structural features of the intrinsically disordered EWS domain in Ewing sarcoma.

Mapping the Structure-Function Relationships of Disordered Oncogenic Transcription Factors Using Transcriptomic Analysis

This experimental protocol provides a stable, cost-effective, and efficient method that combines lncRNA knockdown with colony-forming assays to quantify the effect of lncRNA on osteosarcoma cell proliferation.

Colony Formation Assay Detecting the Proliferative Capacity of LncRNA-knockdown Osteosarcoma Cells

Learn the focus formation assay to evaluate oncogenic potential using retroviral vectors and NIH 3T3 mouse fibroblast cells. This method enables researchers to screen candidate oncogenes for transforming activity.

Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene

Explore More Videos