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Mar 2013

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Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts    

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Cellular transformation is a widely used method to artificially induce cells to form tumours in vivo. Here, we describe the methodology for malignant transformation of mouse embryonic fibroblasts (MEFs) for transplantation into immunodeficient nude mice, as used in Leong et al. (2013). The two-step process involves: 1) down-regulation of Trp53 expression using a short hairpin RNA (shRNA); and 2) overexpression of the oncogenic HRasV12 protein. Reduction of Trp53 expression leads to cell immortalisation, and the subsequent overexpression of oncogenic HRasV12 results in malignant transformation of a cell.

Keywords: Transformation, Mouse embryonic fibroblasts, HRasv12, P53 knockdown

Materials and Reagents

  1. Source of tissue: body of embryonic day 13.5 mouse embryos, harvested fresh from pregnant females
  2. Dulbecco’s Modified Eagle’s Medium (DMEM) (Life Technologies, Gibco®, catalog number: 41965-039 )
  3. Fetal Calf Serum (FCS) (Life Technologies, Gibco®, catalog number: 10437-028 )
  4. Trypsin (Life Technologies, Gibco®, catalog number: 25200056 )
  5. Dulbecco’s Phosphate Buffered Saline (PBS), without Ca2+ and Mg2+ (Life Technologies, Gibco®, catalog number: 14190-144 )
  6. Retroviral supernatant containing LMP-p53.1224 shRNA construct (Dickins et al., 2005)
  7. Retroviral supernatant containing pWZL-HRasV12 cDNA construct (Serrano et al., 1997)
  8. Hygromycin B (Life Technologies, catalog number: 10687-010 )
  9. Puromycin (Sigma-Aldrich, catalog number: P9620-10ML )
  10. Hexadimethrine bromide/Polybrene (Sigma-Aldrich, catalog number: H9268 )
  11. Polybrene (1,000x stock) (see Recipes)


  1. Tissue culture flasks T75 (Greiner Bio-One, catalog number: 658175 )
  2. 10-cm tissue culture dishes (BD Biosciences, Falcon®, catalog number: 353003 )
  3. 21-gauge needles
  4. 5 ml syringes
  5. 37 °C 10% CO2 cell culture incubator
  6. Table-top centrifuge


  1. Retroviral supernatants are prepared as previously described, at a titer of 106 to 107 viral particle per ml of viral supernatant (Pear et al., 1993).
    Note: Do not freeze/thaw supernatant, and use within 6 months.
  2. Primary MEFs are generated from embryonic day 13.5 (E13.5) embryos by passing the embryonic body (excluding head, liver and intestines) through a 21-gauge needle and syringe followed by repeated pipetting into a 10-cm tissue culture dish (1 embryo per dish) in 1 ml of DME medium containing 10% (v/v) FCS (DMEM/FCS). It is not necessary to obtain a single cell suspension at this stage, as trypsinisation at later stages will produce a single cell suspension and excessive manipulation at this stage promotes cell death. Add 9 ml of DMEM/FCS and mix to combine.
  3. Primary MEFs are then incubated in 10% CO2 incubator at 37 °C for 2-3 days undisturbed.
  4. MEFs are washed once in PBS, trypsinised, trypsin inhibited with DMEM/FCS and pelleted at 485 g for 5 minutes.
  5. MEFs are split ~1:2 into a T75 tissue culture flask and incubated in 10% CO2 incubator at 37 °C overnight so that cells are ~60-70% confluent the following day.
  6. On the next morning, aspirate the supernatant and wash once with PBS. Combine the retroviral supernatant containing LMP-p53.1224 shRNA, DMEM/FCS and polybrene using the following recipe:
    Retroviral supernatant          1.5 ml (i.e., ~1:7 dilution)
    DMEM/FCS                          8.5 ml
    Polybrene (1,000x stock)     10 μl (4 μg/ml)
    Total                                     10 ml
  7. After ~7-8 h of infection, repeat step 6, and leave the fresh retroviral supernatant overnight.
  8. On the next day, aspirate the supernatant, wash cells once with PBS, replace with fresh DMEM/FCS, and incubate at 37 °C overnight.
  9. On the following day, replace medium with fresh DMEM/FCS containing 5 μg/ml puromycin (LMP-p53.1224 shRNA construct has a puromycin selectable marker), and leave for 2 days, if not confluent. Otherwise, split as necessary.
  10. At the end of puromycin selection on day 3, cells are washed once with PBS, trypsinised and seeded so that cells are ~60-70% confluent in a T75 flask the following day. Culture cells in DMEM/FCS without puromycin and incubate overnight at 37 °C.
  11. On the next day, repeat steps 6-8, but with retroviral supernatant containing pWZL-HRasV12 cDNA. The two tranductions should be performed sequentially, as suggested, so that p53 knockdown and immortalization precedes HRasV12 overexpression. This ensures the best efficiency of transformation since HRasV12 overexpression with inefficient p53 knockdown results in senescence.
  12. On the following day, replace medium with fresh DMEM/FCS containing 300 μg/ml hygromycin (pWZL-HRasV12 cDNA construct has a hygromycin selectable marker) for 6 days. Replace with fresh hygromycin after 3 days, and split cells when necessary.
  13. At the end of hygromycin selection on day 7, replace with fresh DMEM/FCS without hygromycin.
  14. Passage cells as necessary for another 10-14 days to allow HRasV12 to drive cell proliferation. These transformed cells can now be used for in vitro or in vivo experiments. For example, cells can be injected subcutaneously into the flank of nude mice to assess tumour growth rate in vivo. The cells can be frozen and stored in liquid nitrogen, or can be continuously passaged, however extended passaging will result in additional genetic aberrations based on the knockdown of p53.


  1. 1,000x stock polybrene (4 mg/ml)
    Mix 0.2 g of hexadimethrine bromide with 50 ml Milli Q H2O  
    Filter sterilize (0.22 μm)
    Aliquot and store at -20 °C.


This protocol was previously used and adapted from Leong et al. (2013).


  1. Dickins, R. A., Hemann, M. T., Zilfou, J. T., Simpson, D. R., Ibarra, I., Hannon, G. J. and Lowe, S. W. (2005). Probing tumor phenotypes using stable and regulated synthetic microRNA precursors. Nat Genet 37(11): 1289-1295.
  2. Leong, H. S., Chen, K., Hu, Y., Lee, S., Corbin, J., Pakusch, M., Murphy, J. M., Majewski, I. J., Smyth, G. K., Alexander, W. S., Hilton, D. J. and Blewitt, M. E. (2013). Epigenetic regulator Smchd1 functions as a tumor suppressor. Cancer Res 73(5): 1591-1599.
  3. Pear, W. S., Nolan, G. P., Scott, M. L. and Baltimore, D. (1993). Production of high-titer helper-free retroviruses by transient transfection. Proc Natl Acad Sci U S A 90(18): 8392-8396. 
  4. Serrano, M., Lin, A. W., McCurrach, M. E., Beach, D. and Lowe, S. W. (1997). Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a. Cell 88(5): 593-602.
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
How to cite: Leong, H. S. and Blewitt, M. (2013). Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts. Bio-protocol 3(15): e844. DOI: 10.21769/BioProtoc.844.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

If you have any questions/comments about this protocol, you are highly recommended to post here. We will invite the authors of this protocol as well as some of its users to address your questions/comments. To make it easier for them to help you, you are encouraged to post your data including images for the troubleshooting.

Steven Shen
Duke University
Dear Drs. Huei San Leong and Marnie Blewitt,
After transduction the prepared Retrovirus Mediated Malignant Transformation of MEF to mouse, in wild type created neoplasm and dramatic tumor grow, however, it could not create in a gene Knockout mouse. What is the possibility, or reason and how to resolve the problem?
If shRNA-p53 transfection is a must step in the oncogenic c-myc puro seleated Myc MEFs or oncogenic Ras puro selected Ras MEFs?

Thanks for your comment and help!
9/13/2014 8:56:43 AM Reply
Marnie Blewitt
Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Australia

Dear Steven,

The p53 knockdown is essential prior to oncogenic Ras transduction, as otherwise Ras with cause senescence.

With regards to why your knockout cells don't create a neoplasm, there are a number of possibilities. Were the knockout cells passaged, transduced and selected alongside the controls? In this way, can you be sure they were properly transformed? If so, it is possible that the knockout cells cannot grow in vivo. Potentially this could be because the gene that has been knocked out is downstream of Ras, meaning even with Ras overexpression you don't transform the cells.

9/14/2014 3:54:26 PM Reply

shobhit mishra
6/10/2014 2:48:23 AM Reply
Bio-protocol Editorial Team

Hi shobhit,

We would suggest that your question could be more specific so that it would be answered more efficiently.

Bio-protocol Editorial Team

6/10/2014 1:58:03 PM Reply

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