how to clone the genomic region for coding the mature miRNA - (Dec/30/2007 )
how to clone the genomic region for coding the mature miRNA? Is it ok for me to find the genomic region around the mature miRNA (+300 and -300) by ensemble? I am not sure whether it is the correct way or not?
For cloning and express a microRNA, the pre-cursor miRNA sequence plus about 100 bp upstream and downstream flanking sequences have to be inserted into an expression vector driven by polyII or III promoter. To obtain the genomic sequence for a microRNA, I do a blat search of the UCSC genomic database using pre-miR sequence or do a search using the microRNA name. Once I got the miRNA in the genome browser, I click "DNA" at the top to export the genomic sequence. In the next window, I can specify the length of up- and down-stream sequences to be exported together.
If the precursor is known, I think it would be better to clone it all in the expression vector and let the drosha-dicer machinery process it to give the mature miRNA. If it is not known you could try to mutagenize the precursor of another miRNA, known to work well when overexpressed in your organism, inserting the sequence of the mature miRNA you want to express instead of the mature miRNA naturally present. See Schwab et al, 2006, Plant Cell 18, 1121-1133.
Happy new year,