Retro-X Retroviral Coloning and Expression System (Clontech) HELP!

Scott Fisher scottfis at cyllene.uwa.edu.au
Wed Nov 20 03:12:24 EST 2002


Hi,

I've recently purchased Clontech's "Retro-X" retroviral expression system
kit and am having huge problems trying to get it up a running. Essentially
the problem relates to the pLAPSN (control Lot# 1120667) and pLXSN (cloning
Lot # 1120666) retroviral vectors supplied with the kit. I've performed
transient TFXns using both PT67 and COS7 cells and shown that the pLAPSN
control vector (both our stock and the original sample supplied) is non-
function in the TFXed cells. I included the pSEAP2-Control vector
(non-retroviral) as an extra control and this was able to TFX both cell
lines efficiently enough to detect in a standard SAP (secreted Alk. Phos)
assay. So I know it's not my cells or my TFXn method.

The problems with the pLXSN cloning vector relate to the inability to
propagate enough recombinant plasmid for down stream use (I know its low
copy number, but I'm having to use 2L culture just to get 0.5-1 ug/ml
plasmid). The same inserts were cloned into the pLNCX2 retroviral vector
withot incident which makes me think my host cells, insert purity, ligations
RXn etc are ok.

I have a feeling that the particular batch that these plasmids came from are
faulty. Has anybody else experienced such problems?

I've contacted Clontech and they haven't been able to offer me any
suggestions as to what is going on.

If any one has used this system before (or is currently using it)and could
offer any advice it would be greatly appreciated.

Thanks,

Scott.
scottfis at cyllene.uwa.edu.au






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