Hi everybody. I have a problem trying to overexpress by plasmid the gene of my interest (ffar4 for GPR120). I'm able to infect T293 (they appear totally green), then i filtered and centrifuged the medium that will be used as medium for my Caco2 and LoVo cell lines. After 48/72hrs the control cells are totally green but not the cells with my plasmid. I checked the ratio of the packaging and envelope and everything is fine. I also collected the 293 medium twice to increase the efficiency and nothing seems to work. I don't understand and i'm getting mad.
Thank you in advance
I will appreciate if you may describe your lentiviral vector system in a little details, in terms of helper construct (providing the viral functions in trans), is it the 3 component system?, type of the env being used (typically VSV-G), etc. When you talk about 'green', is it the GFP expression? Associated with what vector? How are you Transfecting? Packaging? Infecting (transducing)?
T293 cells can be easily transfected with lipofectamine. viral based transduction is used for suspension cells and hard to transfect cells. but T293 cells are better transfected using lipofectamine or polyplus transfection reagents.
Muhammad Ayub Khan as our colleague said, i used lipofectamine protocol. The helper constr are PLP1 PLP2 and VSVG. I mentioned green because both my control cells are tagged with EIaF GFP and my plasmid for GPR120 has GFP as well. So basically I have transfected with lipofectamine T293 and their medium is used for Caco2 (colon cancer cell lines) infection. But when I checked for the "green proof" , caco control (so just with GFP) are totally green while the flask with my plasmid (GPR120) has really low amount of infected cells. I can't understand why.
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