Hello, dear researchers. I am interested in a gene in Pombe. The gene contains 2 introns. I want to overexpress this gene in Pombe. Can I amplify and clone the gene by PCR or do I need to do cDNA translation first? Since the gene already belongs to pombe, will the presence of introns cause any problems?
If it is a gene from Pombe then the intron should not be a problem if expressed in the same species.
If you're planning to express it in S. pombe, I would keep the introns. S. pombe should be able to express its own genes, and it may also need the introns for optimal expression levels and expression regulation purposes.
Hello Umut Karatekin ,
what are the size of your gene with and without introns ?
The advantage of using a cDNA without intron is that it reduce the size of the DNA sequence to introduce in your vector backbone and also the final size of your plasmid which would facilitate both cloning and transfection.
The negative side of the cDNA approch is that it could modify how the mRNA is taken care of by your yeast as Carlos Pérez Arques pointed out. A cDNA sequence would produce an mRNA which doesn't undergo splicing and could negatively regulate nuclear export and translation efficiency for example.
Good luck,
Philippe.
As highlighted by Philippe Paget-Bailly the final size of the DNA sequence in your plasmid is something to really consider, since cloning larger fragments is always more challeging and often takes longer and repeated attempts, based on my experience. I have overexpressed several genes without introns and results are satisfying. However, exon junction complexes sometimes are really important in mRNA transport from the nucleus to the cytoplasm and in translation, so this processes can be compromised in intron-lacking constructs.
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