Hi Jochen, I think, the answer to your question is simple. Sorting of GFP positive cells will give you all GFP expressing cells, the transiently transfected cells as well as the stable transfected cells. The problem is, that you will have much more (nearly a factor of 1000) transiently transfected cells. These cells will loose the fluorescence after some days. You have to sort for GFP positive cells after a week again to get now the stable transfected cells. In contrast, if you select the cells using G418, you will kill all cells which are not stable transfected. The difference is, in most cases, that people using a sorter are looking for GFP positive cells after 2 days (to sort a high number of GFP positive (but in most cases transiently transfected) cells) and people using G418 selection have to wait for a week or more until all non-transfected cells have been killed (and get afterwards a higher portion of stable lines). Hope this helps Andreas On 19 Apr 2002, at 14:38, Jochen BARTHS wrote: > > Dear Flow-ers, > > We recently encountered some problems with the sort of transfected cells. > > When we sort GFP+ mouse embryonic stem cells (48 h after Xgen transfection > with a vector expressing the gene of interest and GFP), the sorted positive > cells gradually loose their fluorescence within 3 to 4 days. > > On the other hand the same transfected cells, selected for example in G418, > retain their fluorescence much longer. > Viability etc. of the sorted cells is very good. > > Any ideas, how this phenomenon can be explained, would be appreciated. > (Was there any deleterious effect of cell sorting on long term protein > expression described before?) > > Any proposals how to get around it would be even better. > > Thank you very much in advance. > > Jochen > > Dr. Jochen BARTHS > IGBMC > Service Commun de Cytometrie en Flux > 1, rue Laurent Fries > BP 10142 > F-67404 ILLKIRCH CEDEX > France > Tel.: +33 / (0)3 / 88 65 34 27 or 28 > Fax: +33 / (0)3 / 88 65 32 01 > e-mail: JBarths@IGBMC.u-strasbg.fr ___________________________________________________ Andreas Simm Klinik fuer Herz- und Thoraxchirurgie Universitaet Halle Ernst-Grube-Str. 40 06120 Halle Tel.: +49 (0) 345 557 2647 FAX: +49 (0) 345 557 2782 e-mail: andreas.simm@medizin.uni-halle.de
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:26:07 EST