Retroviral vector and GFP

eugene ekandel at uic.edu
Mon Nov 23 04:19:06 EST 1998


We had an extensive experience with GFP in retroviral vectors, including
LNCX-based ones. CMV works very well in every cell type we tried (a
dozen cell lines of mouse, human and rat origin). Depending on the
precise organization of the construct, cell line and type of humanized
GFP used, positive cells are 50-500 fold brighter then negative control
(single integration). In your case expression may be even higher, since
at 90% transduction rate (provided it was measured accurately) you end
up with multiple proviruses per cell. The likeliest explanations for
your problem is that you are using a non humanized GFP, or an unsuitable
method to detect its expression (inappropriately fixed cells, live cells
in medium with high background fluorescence, suboptimal optics, etc.).

Eugene Kandel
u09577 at uic.edu

Alex Chang wrote:

> Dear netters,
>
> I have a LNCX based retroviral vector which was used for expressing
> GFP. BOSC cells were used for packaging and NIH 3T3 cells used for
> viral titration.
>
> The transient transfection by Superfect of BOSC was great (90%
> efficiency and highy expression level). However, after 48-72 hrs
> infection of 3T3 cells by the packaged retroviral vector, the GFP
> expression level was very low (you can barely see them under
> fluorescent microscope). Eventhough, about 80-90% cells were
> infected.
>
> The retroviral infection was done using the 48 hr harvested viral
> supernatant, which was centrifuged and polybrene mixed (4 ug/ml).
>
> Any clue?
>
> Alex Chang
> Pathology
> University of British Columbia
> alex at cmmt.ubc.ca




More information about the Methods mailing list