This confusion led to anecdotes of IRES failures and a reluctance to use this strategy. A problem that I’ve seen come up is that labs inadvertently incorporated such attenuated versions, expecting equal expression levels of the two proteins and subsequently became frustrated at the results. The rationale for this was that if there was any detectable reporter, then the protein if interest should be present and at higher levels. For example, an attenuated version was produce so that the second protein, the reporter luciferase or GFP, was expressed at a lower level. Labs and companies made subtle changes to tweak the system to adapt the function to different uses. luciferase) to quantitate expression levels in transfected cell lines. IRES sequences where quickly adopted for use in expression vectors, often to express a protein under study along with a reporter (e.g. These sequences where identified in viral genomes where efficient use of limited nucleotide length is valuable. A single RNA is produced but due to the presence of the IRES, a second translational start is possible on the same RNA. ![]() PaeR7I does not recognize the sequence CTCTCGAG.Ĭ A C C T G C ( N ) 4 G T G G A C G ( N ) 4 ( N ) 4Įfficient cleavage requires at least two copies of the PaqCI recognition sequence.IRES sequences are used to express two proteins from a single promoter in an expression construct or a transgenic construct. I-PpoI is a homing endonuclease that can recognize a variety of similar recognition sequences. Prolonged incubation with NdeI may lead to removal of additional nucleotides.Ĭ T C T C T T A A G G T A G C G A G A G A A T T C C A T C G Sticky ends from different Tth111I sites may not be compatible.Įfficient cleavage requires at least two copies of the PluTI recognition sequence.Įfficient cleavage requires at least two copies of the NarI recognition sequence.īsrGI is typically used at 37☌, but is even more active at 60☌.
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