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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+assays+for+Ras+transformation.">Biological assays for Ras transformation.</a> Methods in enzymology. , 255-395 (1995).</li><li>Celis, J. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Cell biology : a laboratory handbook. , 345-352 (2006).</li><li>Raptis, L., Vultur, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neoplastic+transformation+assays.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overexpressed+pp60c-src+can+induce+focus+formation+without+complete+transformation+of+NIH+3T3+cells.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+and+biological+activity+of+human+homologs+of+the+raf/mil+oncogene.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-myc+can+cooperate+with+ras+to+transform+normal+cells+in+culture.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transforming+activity+of+purinergic+receptor+P2Y,+G+protein+coupled,+8+revealed+by+retroviral+expression+screening.">Transforming activity of purinergic receptor P2Y, G protein coupled, 8 revealed by retroviral expression screening.</a> Leukemia &amp; lymphoma. 48, 978-986 (2007).</li><li>Morita, S., Kojima, T., Kitamura, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Plat-E:+an+efficient+and+stable+system+for+transient+packaging+of+retroviruses.">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/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Advanced+mammalian+gene+transfer:+high+titre+retroviral+vectors+with+multiple+drug+selection+markers+and+a+complementary+helper-free+packaging+cell+line.">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. 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J., Wright, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-phase-specific+expression+of+the+Mad3+gene+in+proliferating+and+differentiating+cells.">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. 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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 border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">pBABE-puro vector</td>
			<td style="width:127px;">Addgene</td>
			<td style="width:123px;">Plasmid 1764</td>
			<td style="width:171px;">cloning vector</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Platinum-E Retroviral Packaging Cell Line, Ecotropic</td>
			<td style="width:127px;">Cell Biolabs, Inc.</td>
			<td style="width:123px;">RV-101</td>
			<td>cell line for viral production</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">NIH 3T3 Cell Line murine</td>
			<td style="width:127px;">Sigma-Aldrich</td>
			<td style="width:123px;">93061524</td>
			<td>cell line for focus formation assay</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">10 mL BD Luer-Lok tip syringe</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">309604</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.45 &#956;m Puradisc Syringe Filter</td>
			<td style="width:127px;">Whatman</td>
			<td style="width:123px;">6750-2504</td>
			<td>viral production reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polyethylenimine (PEI)</td>
			<td style="width:127px;">Polysciences, Inc.</td>
			<td style="width:123px;">23966-2</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Polybrene Infection / Transfection Reagent</td>
			<td style="width:127px;">EMD Millipore</td>
			<td style="width:123px;">TR-1003-G</td>
			<td>cell transfection reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Crystal Violet</td>
			<td style="width:127px;">Fisher Scientific</td>
			<td style="width:123px;">C581-25</td>
			<td>cell stain reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Plasmid Plus Midi Kit</td>
			<td style="width:127px;">QIAGEN</td>
			<td style="width:123px;">12945</td>
			<td>plasmid purification</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">BD Falcon Tissue Culture Dishes</td>
			<td style="width:127px;">BD Biosciences</td>
			<td style="width:123px;">353003</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Dulbecco&#39;s Modified Eagle Medium (DMEM)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">11995-065</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">0.05% Trypsin-EDTA</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">25300-054</td>
			<td>cell culture supplies</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Opti-MEM I Reduced Serum Medium</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">31985-062</td>
			<td>cell culture media</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:413px;">Fetal Bovine Serum (FBS)</td>
			<td style="width:127px;">Gibco</td>
			<td style="width:123px;">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

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...

Watch this Scientific Journal Video about Focus Formation: A Cell-based Assay to Determine the Oncogenic Potential of a Gene at JoVE.com

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

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 ...

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27.6K Views.  University of California, Davis. The Focus Formation Assay provides a straightforward method to assess the transforming potential of a candidate oncogene.