If the protein in question is inducible by activation of a signaling pathway, one can use this as a reference to achieve "overexpression". One needs to consider other factors also such as appropriate sub-cellular localization in "overexpressed" cells.

Hi, Dr. Choubey, we are looking at a G protein alpha subunit, based on your suggestion, should I check this G alpha's activity in response to growth factors first? Thank you

Do you have access for the health volunteer and patient samples for the disease in question? If not, is there an animal model for the disease that you may compare between health and diseased tissue?

Hi Dr. Tseng, so far, expression of this protein has not been found elevated in any diseases. And no animal models were used for overexpression. 

That is interesting. Is there anything altered in the diseases, ie.  localization, functional activity, or protein structure, either increase or decrease? If only overexpression of the protein in primary HUVEC is interest, there are different methods that I would use for analysis, using mock treated primary HUVEC as control, to show whether the overexpression affects the cellular functions. About the "proper" level of overexpression is depended on the function of interest. 

Dr. Tseng, activity of this G alpha was found elevated in melanoma, due to mutation, but not expression level. We do found siRNA can decrease growth factors-stimulated migration in HUVECs.

And our current idea of following experiments are doing dose-dependent experiments of different MOI of adenovirus and to see at what MOI growth factors-induced migration will be changed. Just want to reach out to see if there are better ideas.

Thank you very much for responding.