Amersham Pharmacia Biotech). Aliquots stored at -20°C.
No. B1508; Sigma). Aliquots stored at -80°C.
xylene (2:1, TMPD; Sigma).
counter (Packard, Meriden, CT, USA).
We use CAT activity to normalize for luciferase expression when the site of injection produces mean values with intra- and interassay variability of more than 15%. In such conditions, it is difficult to obtain statistically valid results without normalizing for transfection efficiency with a constituitively active construct (e.g., CMV-CAT). 1. Before using a co-injected ubiquitously expressed gene (CMV-CAT) to normalize for expression from a physiologically regulated transgene, it is appropriate to validate this approach by quantifying the correlation between the expression of two constituitively expressed genes co-injected into the same brain area. For this, two plasmids (0.35 μg pCMV-luc and 0.15 μg pCMV-CAT in 2 μL 5% glucose) are complexed with PEI 22 kDa (4 eq) and co-injected into brain area targeted. After 18 hours, mice are anesthetized, decapitated, and brains removed for luciferase and CAT assays. 2. For CAT assay, transfer a 50-μL supernatant aliquot to a 1.5-mL polypropylene tube, keep the tube on ice before adding 40 μL of 0.25 M Tris-HCl buffer (pH 7.5). Start reaction by adding 10 μL of mix solution of butyryl- CoA (0.53 mM; Sigma) and [14C] chloramphenicol (0.01 mM, 1.85 kBq per tube; Amersham Pharmacia Biotech). After 3 to 5 seconds of vortex mixing, incubate for 1 hour at 37°C. Then, stop the reaction by adding 200 μL of TMPD/xylene solution (2:1). Vortex mix for 20 seconds and place tube on ice. To separate products, centrifuge for 5 minutes at 4°C (11 000× g), remove 150 μL of supernatant, and quantify products in a scintillation counter (Amersham Pharmacia Biotech, Piscataway, NJ, USA). 3. Assay luciferase on other sample of supernatant (see above). 4. Plot luciferase against CAT values from the same sample. Correlation should be significant.
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