I am working on human variants of a certain gene. Most of the time I care about non-synonymous substitutions since they are easy to reproduce and work with. But I got interested in one synonymous substitution which seems to be generating a new splice site inside of one of the last exomes. I wanted to re-create that substitution in the plasmid that I normally use for expression of my gene. I expect to truncate the protein but am I right? Did anyone introduce a splice site in the gene (cDNA) that is cloned into the plasmid?
Michal
HI,
thanks Pia.
this is not a splice site per se, exon/intron junction. It is a mutation that generates donor site within the exon. I do not expect to successfully splice it and generate shorter protein but to induce splicing by introducing mutation and as a consequence truncate protein and/or diminish its expression due to mRNA instability.
Michal
Does this mutation induce a 5' splice donor site or a 3' splice acceptor site? Assuming it's a splice donor site, and this splice site is present in your gene that is within your plasmid, then you will likely see no protein expression of your transgene, because the mRNA will likely not have the poly A signal sequence. You might splice out a large chunk of RNA if there is a cryptic splice acceptor site downstream of the donor site within your plasmid. However to know what happens in vivo, Pia is correct, you'd really need to know what is happening within the context of the genome. Presumably you could predict this, by finding the first 3' splice acceptor site that is downstream of this mutant 5' splice donor site.
I would first ensure you understand how when and where splicing occurs and what features need to be present within a sequence before it is spliced. It seems to me you don't understand the fundamental basis of splicing. cDNA will not be spliced nor will it necessarily make the mRNA more unstable. However, what may change the amount of protein is if the codon it generates is rarely used in the expression host. The limiting factor would be the amount of tRNA available for that codon. You would need to check the codon usage tables for the host.
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