psPAX2 / pCMV-VSV-G / pLJM1-EGFP ===> when to see the EGFP???

[owl.crow]

Dear all,

I am using the LentiCRISPRv2 plasmid to try to KO a protein of interest. At the moment I am just using the CONTROL empty backbone vector pLJM1-EGFP along with the other two (see below) to optimise my CaCl2 trasnfection.

  pLJM1-EGFP/ psPAX2 /  pCMV-VSV-G   (4:3:2)


After transfection of HEK293 i CAN NOT see any EGFP.  My question is:   I will see the EGFP before being packaged (ie. after HEK293 trasnfection) or  in the final cells which are infected with the virus (i.e. my final target cells).

Thank you very much for your invaluable help!

[Neville Sanjana]

Hi,

I haven't used the exact lentiviral transfer plasmid you mention but for all control transfer plasmids I've used (e.g. lenti-CMV-EGFP or similar), you should see EGFP in both the HEK293 packaging cells and the infected target cells.

CaCl2 transfection is notorious for being very dependent on getting exactly the right pH for the HeBS buffer.... you might want to try a lipofection reagent (e.g. Lipofectamine 2000), which is a much easier protocol. If you need to do CaCl2, I've found it helpful to make a few different pH versions of HeBS and empirically test the transfection efficiency.

Best,

Neville

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