Q. regarding rat liver NADPH-cytochrome P450 reductase

Steven Goldberg goldberg at bms.com
Thu Mar 6 13:29:55 EST 1997


I have been trying to isolate the rat liver NADPH-cytochrome P450
reductase gene to no avail.  Since the sequence of this gene is known, I
created primers for PCR to amplify the entire gene using cDNA purchased
from Clontech or from a cDNA library from Stratagene as template.  In
neither case could I get even a hint of a band despite numerous attempts
at optimization.  I then tried making new primers to different parts of
the gene with the same (negative) result.  Ditto with hybridization using
labeled oligonucleotide.

Looking at the original paper about the cloning of this gene I noted that
the rats had been injected with phenobarbitol to presumably induce the
mRNA encoding the reductase for cDNA preparation.  The cDNA and library I
have been using were made from livers of normal rats.  Could this be the
reason I am not seeing any amplification or hybridization?  Also, if
anyone has a source of this gene or cDNA prepared from livers
phenobarbito-induced rats I would be grateful to hear from you.

Thanks for your assistance.


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