Well, loosing fluorescence would depend on when the gene is excised, how stable is the remaining mRNA, and how fast the remaining protein is degraded/diluted. By lipofection, it would take about one day for the Cre to be expressed and then it should be immediately active. EGFP has a half-life of about one day and the cells still have mRNA, so they will be green for some more days. Cell division of course dilutes both residual RNA and protein, so that should speed it up, so how fast are your cells dividing?. Another thing of course is how efficient the Cre transfection was. If you have only a minority of cells transfected, then you won't see much. If you have a flow cytometer around, you could check for more subtle changes in fluorescence than by eye.

Hope that helps.

Have you done a quick 'transient' assay with the Cre-expressing vector and the lox-Promoter-GFP-lox construct to make sure that site-specific recombination is actually happening? To ensure that your constructs are ok. This can be done by co-transfect cells with these two plasmids, and check for Cre-mediated excision with PCR.

Christian, Thank you for the comment. You are right. Transfection efficiency and cell doubling time are my concerns as well. Transfection can be as low as 30% with Xfect or lipofection for this cell line and doubling time is usually 3-4 days. We are thinking about creating nonintegrative lentivirus vector and use it to boost up the efficiency. 

Yau, I have not tested it. But it is a good idea. I will try it! Thank you! 

This is a super old post. We ended up using AAV-Cre, The knockout efficiency is >90% in 1 week.

Hi Danny Ko,

Thanks for updating us. Appreciate that! Most people just don't come back after discussion and give an update

Three questions:

1. What is this AAV-Cre special about? I am in plant field, but I am interested to know.

2. After 2 or 3 days after transfection, did you still see green fluorescence in many cells?

3. What did you mean by "creating nonintegrative lentivirus vector and use it to boost up the efficiency" (in your previous answer)?

Hi Yuan, 

1. Adeno-associated virus (AAV) infects our human fibroblast with high efficiency and expression lasts longer than transfection method, and it rarely integrates into the genome. So I think AAV is ideal for gene delivery when working with hard-to-transfect cell lines.

2. I remember seeing GFP+ cells 2-3 days after infection, but almost none after 7 days. 

3.  I meant using that vector to package integrase deficient lentivirus (IDLV). Because lentivirus is efficient in infecting mammalian cells, so I thought  IDLV can also be efficient in delivering cre  into the cell without integrating it to the genome.

Hopefully I have answered your questions.  Let me know if you others. 

Thank you for your response.

What make AAV rarely integrate into the genome?

So your experiment is kind like 'transient' (without the foreign DNA integrate into the genome), just transiently express the cre gene. In plants, we also prefer to transiently express a cre gene (by gene bombardment or PEG-mediated genetically transformation), so we don't have to segregate the cre gene away (if it integrates into a genome) in the future. 

I'm not an expert on virus.But I think that unlike lentivirus, AAV vectors don't encode proteins required for integration. 

Thanks for replying, Danny Ko.

Thank you Danny, I am going to perform the AAV-Cre for the mice in vitro cell culture. I am wondering do you select any antibiotic marker for AAV-Cre during the one week waiting time? Or just assume all the cells will express Cre? My AAV-Cre has eGFP marker, so maybe I can monitor it by looking at the fluoresence?