欢迎您, 登录 | 注册

首页 | English

X
加载中

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.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts
逆转录病毒介导小鼠胚胎成纤维细胞的恶性转化

癌症生物学 > 通用技术 > 细胞生物学试验 > 细胞转化
作者: Huei San Leong
Huei San LeongAffiliation: Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
Bio-protocol author page: a725
 and Marnie Blewitt
Marnie BlewittAffiliation: Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Melbourne, Australia
For correspondence: blewitt@wehi.edu.au
Bio-protocol author page: a724
Vol 3, Iss 15, 8/5/2013, 2783 views, 2 Q&A, How to cite
DOI: http://dx.doi.org/10.21769/BioProtoc.844

[Abstract] 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(转型), Mouse embryonic fibroblasts(小鼠胚胎成纤维细胞), HRasv12(hrasv12), P53 knockdown(p53基因敲除)

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)

Equipment

  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

Procedure

  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.

Recipes

  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.

Acknowledgments

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

References

  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.


How to cite this protocol: 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; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
9/13/2014 8:56:43 AM  

Steven Shen
Duke University

Dear Drs. Huei San Leong and Marnie Blewitt,
Hello!
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!

9/14/2014 3:54:26 PM  

Marnie Blewitt (Author)
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.

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

6/10/2014 2:48:23 AM  

shobhit mishra
TMU

WHAT WILL BE THE EXPECTED ESTIMATE TO DO THIS EXPERIMENT IN INDIA

6/10/2014 1:58:03 PM  

Bio-protocol Editorial Team
bio-protocol.org

Hi shobhit,

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

Thanks,
Bio-protocol Editorial Team

Reply

Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

Login | Register

分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook