Luciferase expression variability

Graham Atherton grggta at picr.cr.man.ac.uk
Wed Sep 21 05:22:57 EST 1994


We have just completed and experiment which consisted of the following:
1) B-gal reporter 
2) CAT-reporter
3) Luciferase reporter

All should be constitutively expressed and were co-electroporated into 
seperate aliquots of cell five times. The one variable between the five
electroporations was a fourth plasmid expressing sense, antisense, empty
vector or no DNA control. Each aliquot got a different variant of this
fourth plasmid.

Assaying for B-gal (% blue cells) and CAT showed approx 2-3 fold variation
between the cell types, probably telling us that there is a small amount
of variation between the quality of our plasmid preps causing variation
of DNA uptake. The luciferase (the third internal control) however gave
1000 -fold variation between the five aliquots - wildly exagerating the 
CAT and B-gal results though retaining the same pattern of variabilty -
the samples that give low reading for CAT and Bgal also gave low reading
for luciferase and so on.
Has anyone else seen such enhancment of apparent uptake of DNA by the use
of luciferase as an internal control? Is there any way to control it -
we have tried protease inhibitors to try to prevent protease attacking
the luciferase with limited success.
Could this effect explain why I spent a very long time trying to get
an induction of a reporter using CAT while our competitors had no(?)
problems when using luciferase?
NB it is unlikely that the gene we are expressing could have caused this
effect, it is dependant on procaryotic transcription factors for expression.
All three internal control have different enhancer/promoters.

Yours frustrated
Graham Atherton
grggta at picr.cr.man.ac.uk



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