Huei San San Leong —BIO-PROTOCOL

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Marnie Blewitt Molecular Medicine Division, The Walter and Eliza Hall Institute of Medical Research, Australia
1 protocol

Reviewer

Huei San San Leong

The Walter and Eliza Hall Institute of Medical Research

Research focus

Cancer biology

1 Author merit
Reviewer merit
Editor merit

Education

Ph.D. in Cancer Epigenetics, Walter and Eliza Hall Institute of Medical Research, University of Melbourne, Australia, 2012

Current position

Post-doctoral scientist, Peter MacCallum Cancer Centre, Melbourne, Australia

Publications

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.

1 Protocol published

Retrovirus Mediated Malignant Transformation of Mouse Embryonic Fibroblasts

Authors: Huei San Leong and Marnie Blewitt, date: 08/05/2013, view: 5968, Q&A: 2

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 ...

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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!

9/13/2014 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.

2014-09-14 15:54:26