Overexpression

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aureliegineste
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Overexpression

Post by aureliegineste » Wed Sep 04, 2013 7:48 am

Hi everyone !
I need some help. I would like to overexpress a gene (zfp36) in bone marrow derived-macrophages culture (BMDM). I started my PhD few days ago and I don't have a lot of informations about this technical procedure. So I ask you some questions:
- Do you have some advice about the vector providers (I'm in France)?
- When I will make the transfection, I will need to select the transfected cells : is it better to use fluorescent proteins (as GFP) or antibiotics ?
- A lot of protocol are designed for cell line macrophage but not for primary cells as BMDM. Is everyone know if there are some differences between the protocols ?

Thank you so much for your help
Aurélie

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JackBean
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Post by JackBean » Thu Sep 05, 2013 11:41 am

Sorry, I have no experience with cell cultures as yours, but from what I can say:
Your location doesn't really matter for ordering plasmids, you will probably buy it from some big company anyway. Just find the best vector for you (including selection).
GFP is IMHO suitable for single cell selection (or other work after the selection via other means). On the other hand with antibiotics you can easily select cells with your plasmid. Or you can use heterotrophy for the selection (and I can imagine that bone marrow macrophages are heterotrophic at least for something).
http://www.biolib.cz/en/main/

Cis or trans? That's what matters.

OliviaZ
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Post by OliviaZ » Tue Apr 08, 2014 10:45 am

Dear Aurelie,

we are a young biotech company from Germany and have developed a new kind of transfection reagent called Viromer.
Its a synthetic polymer , zero charged with an endosome escape mechanism. The advantages compared to other transfection reagents are: high transfection efficiency in hard to transfect cells like Primary macrophages as well as in standard cells, you can work with complete media (including FCS) without changing media, high reproducibility because Viromers form stable particles of 300-400nm in buffer.
If you are interested I may send a free sample to you. We provide two transfection reagents: for siRNA (Viromer BLUE and GREEN) and for plasmids (Viromer RED and YELLOW).

Please write to: [email protected] (email address of our company).

Best wishes,

Olivia

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