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luciferase vector construction - (Mar/10/2008 )

dear all:
I have a receptor protein which has splice variant in the cell. I want to monitor the splicing thing in the cell. so i want to build a construct with the promoter and the gene and the gene itself and the luciferase. I have the plasmid of the gene with its promoter. can i just cut the Luciferase ORF from TOP plasmid ( or other luciferase reporting plasmid) and subclone it into my original gene vector? will the luciferase work by this way? because it will become a fusion protein.
Thank you for any input.
Niu

-Niu-

QUOTE (Niu @ Mar 10 2008, 04:29 PM)
dear all:
I have a receptor protein which has splice variant in the cell. I want to monitor the splicing thing in the cell. so i want to build a construct with the promoter and the gene and the gene itself and the luciferase. I have the plasmid of the gene with its promoter. can i just cut the Luciferase ORF from TOP plasmid ( or other luciferase reporting plasmid) and subclone it into my original gene vector? will the luciferase work by this way? because it will become a fusion protein.
Thank you for any input.
Niu


You need to make sure that luciferase is in frame with the transgene without a stop codon between the 2 genes.

-scolix-

thanks.
what i am concerned is if luciferase assay will work with this fusion protein.
Bing


QUOTE (scolix @ Mar 11 2008, 11:19 AM)
QUOTE (Niu @ Mar 10 2008, 04:29 PM)
dear all:
I have a receptor protein which has splice variant in the cell. I want to monitor the splicing thing in the cell. so i want to build a construct with the promoter and the gene and the gene itself and the luciferase. I have the plasmid of the gene with its promoter. can i just cut the Luciferase ORF from TOP plasmid ( or other luciferase reporting plasmid) and subclone it into my original gene vector? will the luciferase work by this way? because it will become a fusion protein.
Thank you for any input.
Niu


You need to make sure that luciferase is in frame with the transgene without a stop codon between the 2 genes.

-Niu-

You need to try it out. Fusion proteins behave differently. So its difficult to predict protein expression and activity.

Other option is to have a bicistronic vector, that is having 2 expression cassettes. One would be the gene of interest and other luciferase. This should work without any problem.

-scolix-