Over-expresssion of miRNAs? - (Nov/09/2007 )
I see lots of questions about doing miRNA knockdowns but is there a way to over-express them?
-katjo-
you can do this in several ways:
1) Transfect pre-miRNA or mature microRNA
2) Clone the microRNA gene into an expression vector such as those used for expressing shRNA. For this purpose, you can pcr amplify the microRNA gene and some of its flanking sequences.
-pcrman-
Yeah, i'm working on a cloning protocol now for sequencing but I mean in cells or in the animal...to see phenotypic affects.....
-katjo-
QUOTE (pcrman @ Nov 9 2007, 02:20 PM)
you can do this in several ways:
1) Transfect pre-miRNA or mature microRNA
2) Clone the microRNA gene into an expression vector such as those used for expressing shRNA. For this purpose, you can pcr amplify the microRNA gene and some of its flanking sequences.
1) Transfect pre-miRNA or mature microRNA
2) Clone the microRNA gene into an expression vector such as those used for expressing shRNA. For this purpose, you can pcr amplify the microRNA gene and some of its flanking sequences.
Then you can generate miRNA transgeic animals ,there already have been sevral papers about transgeic mice
-zany-
I guess chances are very low to see a phenotype change in transgenic mouse. Even a gene is a validated target of the miRNA, it may be regulated by multiple miRNAs. By overexpressing this miRNA, it is hard to predict whether the gene expression will change or not.
-pcrman-
QUOTE (pcrman @ Nov 30 2007, 09:57 PM)
I guess chances are very low to see a phenotype change in transgenic mouse. Even a gene is a validated target of the miRNA, it may be regulated by multiple miRNAs. By overexpressing this miRNA, it is hard to predict whether the gene expression will change or not.
I'm a afraid that some miRNA transgenic mice show very severe phenotypes ,ie mMIR-24 transgenic mice
-zany-