Cloning - intronic sequences needed? - (Apr/20/2006 )
Hi
When I want to clone a gene into a expression vector , I usually amplify it by PCR using a cDNA library as DNA template.
As a result of that I amplify the gene coding sequence, therefore the intronic regions will not be cloned.
Can anybody please tell me if this is common pratice for cloning genes?
or do people use genomic DNA as template for performing the PCR?
What would be the difference in terms of protein expression having the genomic sequence or coding sequence of a gene cloned in a expression vector?
Thanks
if you want to express a full protein from your gene of interest, then use cDNA.
Your genomic sequence for the gene will be huge!!! and in addition to being a nightmare to clone, you need to be sure that the recipient cell (line) will be able to effectively process the produced pre-mRNA into the correct splice form.
It is common practice as far as I know, but I believe including/not including introns when cloning for expression depends on your needs.
Indeed, you need to be sure your recipient cells will be able to effectively process the RNA, but on the other hand ,if they do, chances are that your expression levels will be higher due to the presence of introns (the splicing machinery supposedly stabilizes some other enzymes involved in translation leading to a higher protein yield per RNA copy...).
Your genomic sequence for the gene will be huge!!! and in addition to being a nightmare to clone, you need to be sure that the recipient cell (line) will be able to effectively process the produced pre-mRNA into the correct splice form.
Yes, you are absolutely right. Cloning the genomic sequence of this gene would mean cloning 24kb while the cDNA is only 580 bp.
You say to have in account the cell line used to express the protein of interest. What do you mean?
Are you refering for instance to cases where a human protein is being expressed in E.coli?
E.Coli will not be able to process the mRNA?
Can you explain or tell me where I can get more information on this please?
Thanks
If you want to express your protein in e. coli, you better NOT include the introns. Prokaryotics do not have introns and cannot process them... Also, you need to be sure that e. coli can perform all the post-translational modifications (like glycosylations etc.) that sometimes happen with proteins in human cells. Check your protein of interest first!
Further: one cell line is not the other. A T-cell line is completely different from skin cells, which is again completely different from cells of the central nervous system etc... You also can not really predict any secondary effects of protein expression in a cell line (interactions with other proteins etc...).