As an example, the pEF vector available from Sigma, as well as most EF1 vectors on addgene have a 1172 bp EF1a promoter sequence.
The SystemBio pCDH vectors have a smaller 545bp EF1a promoter sequence.
I'm looking to clone the EF1a promoter into my expression system.
I'm unsure which sequence to clone.
Hi Martin
The core EF1a promoter is only about 200-300bp in size. The 1172bp version actually contains an intronic sequence, which is occasionally spliced out. This extra sequence gives a higher level of expression than the core EF1a promoter alone.
Which expression system will you be using? If it is lentivirus, then the intron might not always be retained throughout the production process.
All the best
John
Answering my own question:
Ive realised that the pCDH vectors use a composite promoter; hEF1a-HTLV
http://discovery.ucl.ac.uk/20480/1/20480.pdf
"hEF1a-HTLV promoter is a composite promoter comprising the human Elongation Factor-1α (EF-1α) core promoter and the R segment and part of the U5 sequence (R-U5’) of the Human T-Cell Leukemia Virus (HTLV) Type 1 Long Terminal Repeat. The EF-1α promoter exhibits a strong activity and yields long lasting expression of a transgene in vivo. The R-U5’ has been coupled to the EF-1α core promoter to enhance stability of RNA"
Hi Martin
The core EF1a promoter is only about 200-300bp in size. The 1172bp version actually contains an intronic sequence, which is occasionally spliced out. This extra sequence gives a higher level of expression than the core EF1a promoter alone.
Which expression system will you be using? If it is lentivirus, then the intron might not always be retained throughout the production process.
All the best
John
I'm inserting this promoter into my pcDNA expression system as I have found that high expression of my GOI leads to silencing of the CMV promoter. I confirmed this with 5-aza, which turned expression back on.
Do you know if the EF1a-HTLV composite promoter is prone to methylation like the CMV?
Hi Martin,
In human T cells EF1a (1.2kb version) is a strong and stable promoter (Michael C. Milone, Molecular Therapy, 2009). If it is not too big for your expression plasmid, it might be a safer choice.
Hi John. No, translation start site is downstream of the intron in the native gene, so should not affect GOI frame. if you include a strong Kozak consensus sequence adjacent to the ATG start codon in your GOI, then all should be good.
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