Fusion of reporter genes - (Apr/08/2010 )
Hi Everyone;
I am working with a plant and I want to use of Gus and GFP together to determine the promoter and protein expression and localization.
I would mention that I didnt use of my gene promoter, I used of another promoter to overexpress my gene.
Now the question is how can I fuse 2 reporter gene to get the best result. which one at first? Should I consider any differences between the gene + fusion reporters construct and promoter + fusion reporters construct?
Has anybody done this before?
Any comment and suggestion is greatly appreciated.
Regards,
Geneticlover
-geneticlover-
geneticlover on Apr 8 2010, 12:50 AM said:
Hi Everyone;
I am working with a plant and I want to use of Gus and GFP together to determine the promoter and protein expression and localization.
I would mention that I didnt use of my gene promoter, I used of another promoter to overexpress my gene.
Now the question is how can I fuse 2 reporter gene to get the best result. which one at first? Should I consider any differences between the gene + fusion reporters construct and promoter + fusion reporters construct?
Has anybody done this before?
Any comment and suggestion is greatly appreciated.
Regards,
Geneticlover
I am working with a plant and I want to use of Gus and GFP together to determine the promoter and protein expression and localization.
I would mention that I didnt use of my gene promoter, I used of another promoter to overexpress my gene.
Now the question is how can I fuse 2 reporter gene to get the best result. which one at first? Should I consider any differences between the gene + fusion reporters construct and promoter + fusion reporters construct?
Has anybody done this before?
Any comment and suggestion is greatly appreciated.
Regards,
Geneticlover
Hello,
sounds interesting!
Usally one creates independent constructs with GFP or GUS - so what is your reason for combining both besides reducing construct number and a no-risk-no-fun-mentality?
Some have done this before
e.g. Nadeau & Sack (2002). Control of stomatal distribution on the Arabidopsis leaf surface. Science.
they used a promoter:GUS-GFP fusion, but only promoter:protein-GFP fusions no promoter:protein-GUS-GFP fusions
Have you any experience with these reporter systems?
Because depending on your level of knowledge, I would add something like "Mind stop codons and reading frame!" or not.
Other points to think about would be the cloning system you use (Gateway?) including the vectors and the size of the gene you want to clone, depending on these points and others there could be very fast much more risk than fun - starting right away with creating your constructs!
-SAnna-
SAnna on Apr 12 2010, 11:18 AM said:
geneticlover on Apr 8 2010, 12:50 AM said:
Hi Everyone;
I am working with a plant and I want to use of Gus and GFP together to determine the promoter and protein expression and localization.
I would mention that I didnt use of my gene promoter, I used of another promoter to overexpress my gene.
Now the question is how can I fuse 2 reporter gene to get the best result. which one at first? Should I consider any differences between the gene + fusion reporters construct and promoter + fusion reporters construct?
Has anybody done this before?
Any comment and suggestion is greatly appreciated.
Regards,
Geneticlover
I am working with a plant and I want to use of Gus and GFP together to determine the promoter and protein expression and localization.
I would mention that I didnt use of my gene promoter, I used of another promoter to overexpress my gene.
Now the question is how can I fuse 2 reporter gene to get the best result. which one at first? Should I consider any differences between the gene + fusion reporters construct and promoter + fusion reporters construct?
Has anybody done this before?
Any comment and suggestion is greatly appreciated.
Regards,
Geneticlover
Hello,
sounds interesting!
Usally one creates independent constructs with GFP or GUS - so what is your reason for combining both besides reducing construct number and a no-risk-no-fun-mentality?
Some have done this before
e.g. Nadeau & Sack (2002). Control of stomatal distribution on the Arabidopsis leaf surface. Science.
they used a promoter:GUS-GFP fusion, but only promoter:protein-GFP fusions no promoter:protein-GUS-GFP fusions
Have you any experience with these reporter systems?
Because depending on your level of knowledge, I would add something like "Mind stop codons and reading frame!" or not.
Other points to think about would be the cloning system you use (Gateway?) including the vectors and the size of the gene you want to clone, depending on these points and others there could be very fast much more risk than fun - starting right away with creating your constructs!
Hi SAnna,
Thank you for your reply.
Well, The first reason, as you mentioned, is decreasing the construct, because I have different promoter and gene combination and the second point is since I am working with a palnt which is not easy going like Arabidopsis
and expression of GFP is not convincing in it, I just decided to change the system abit to see if it improves or not.The gene is around 1.8 kb and promoter is 3.2 kb !!!
The current construct including the gene and promoter will be in pTKan vector but I should talk with my supervisor to see what will be the final vector.
Is it necessory that we use of a vector which contains Gus ,or, and GFP or we can introduce them to our vector?
Of course it will be great if you can give me some advice about infame story and other things which you think I should consider, I am not expert in this area and I didnt work with GUS and GFP before.
Any advice, comment, criticise and ... is greatly appreciated
-geneticlover-