Hi All,
I was thinking about in translation part, stop code will stop translation, and ATG will start translation, so what if I make a plasmid, it has promoter, then a cDNA start with ATG and add stop code after it, then add another cDNA which is also start with ATG and has stop code, then SV40-polyA seq. So the plasmid will become like:
Promoter-ATG-DNA1-stop code-ATG-DNA2-stop code-SV40pplyA.
will both of them be translation into protein?
As I feel, the promoter will start transmission and all of the seq from first ATG until SV40-polyA will be trasnmission, then 2 cDNA will both translation as both of them has ATG part and stop code. But I found online that people use 2A or IRES seq to separate 2 cDNA. So I got totally confulsed.
Anyone help?
Hi Monica,
You absolutely need an IRES (internal ribosome entry site) in between your two gene constructs because after the stop codon on the first cDNA is released from the ribosome. Note that usually the second gene after the IRES is translated less efficiently respect to the first.
Hi,
I agree with my colleague Piero. You need IRES in between the two cDNAs to be able to translate them in eukaryotic cells.
Dear Monica
As previous responders have said, if you make a bi- (or multicystronic) construct, you need a way of allowing the ribosome to commence translation of the second (and subsequent) open reading frames. Using and internal ribosome entry site downstream of the previous stop codon is one way of doing it, but rarely are equimolar levels of protein expression produced from the different open reading frames. An increasingly adopted solution to this problem is to introduce "self-cleaving" 2A peptides into multi-cistronic constructs which are smaller than IRES elements and usually result in equimolar levels of multiple gene products from the same mRNA. Instead of using a translational stop codon, these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at the C-terminus of a 2A element, when translating a single open reading frame encoding two or more proteins, leading to separation between the end of the 2A sequence and the next peptide.
It can get quite confusing trying to tease out the correct approach from the primary literature, but there are a few good ‘guides’ available, such as the following, which give an overview of the alternatives and help you to choose the solution best suited to your needs. The good thing is that proven multi-cistronic cloning vectors are becoming available to purchase, so that you can avoid the uncertainties inherent in constructing something from scratch. I’ve also added a reference which shows the "self-cleaving" 2A peptide solution in action.
I hope that helps!
Best wishes
Steve Morley
Addgene's Blog / Post - Plasmids 101: Multicistronic Vectors
Posted by Melina Fan on Sep 9, 2014 4:20:00 PM
http://blog.addgene.org/plasmids-101-multicistronic-vectors
Multiple gene products from a single vector: ‘self-cleaving’ 2A peptides
Radcliffe PA and Mitrophanous KA
Gene Therapy (2004) 11, 1673–1674. doi:10.1038/sj.gt.3302361
http://www.nature.com/gt/journal/v11/n23/full/3302361a.html
Fucci2a: a bicistronic cell cycle reporter that allows Cre mediated tissue specific expression in mice.
Mort RL, Ford MJ, Sakaue-Sawano A, Lindstrom NO, Casadio A, Douglas AT, Keighren MA, Hohenstein P, Miyawaki A, Jackson IJ.
Cell Cycle. 2014;13(17):2681-96. doi: 10.4161/15384101.2015.945381.
http://www.ncbi.nlm.nih.gov/pubmed/25486356
Hi,
If I want to construct 3 genes together as polycistronic mRNA with P2A and say it is like this:
Synthetic Promoter-cDNA1-P2A-cDNA2-P2A-cDNA3-Stop codon
I am using a synthetic vaccinia virus promoter.
The doubt is :
is it necessary for an ATG for cDNA1?
Then, after the P2A sequence, is an ATG required for cDNA2 and cDNA3?
Dr di stasi has answered correctly. The point about using the short "self-cleaving" 2A peptide is that it is a clever way of synthesising two or more proteins from a single mRNA open reading frame. The 2A peptide coding element causes the ribosome to miss out synthesis of a peptide bond, thus cleaving the protein product; however, the ribosome remains attached to the mRNA and thus does not require a 're-entry' sequence and continues to synthesis new protein until it meets a stop codon. In addition to making two or more proteins at roughly equimolar levels, a further advantage is that second (and subsequent) protein(s) in the series do not necessarily require a contextualised ATG (Met) codon and can just be synthesised as mature protein within the cell. However, if it was required that the second protein was to be exported or directed elsewhere in the cell, then a suitable extrinsic or intrinsic 'signal' sequence would also need to be included.
Key considerations:
Use a strong ubiquitous or tissue-specific gene promoter incorporating an appropriately contextualised high efficiency ATG start codon (e.g. for eukaryotic expression, including a transcription start site and TATAAA box sequence located 25-35 base pairs before the transcription start). A good example of this is the CAG promoter cassette mentioned above in Mort et al., Cell Cycle. 2014;13(17):2681-96. doi: 10.4161/15384101.2015.945381.
When using a synthetic promoter, it may be necessary to add and ATG codon to the cDNA to be expressed and to ensure that the appropriate transcriptions start elements are engineered into the final construct.
Also, while STOP codons for the first and intermediate coding sequences should be replaced with the 2A peptide coding element, the final protein in the series will require and in-frame STOP codon. The the ideal constrict should be something like:
Synthetic Promoter-TATAAA-ATG -cDNA1-P2A-cDNA2-P2A-cDNA3-Stop codon
I would strongly recommend that the references quoted above and also more recent publications form the literature should be consulted to become fluent in design of these types of constructs.
Finally, the short "self-cleaving" 2A peptide strategy is aimed at making two or more separate proteins in the same cell at roughly equimolar levels. This strategy is clearly not appropriate where different levels of protein expression are desired or where a fusion protein is required, e.g. for using a fluorescent label to follow the fate of a protein within a cell.
I hope that helps
Steve Morley
- Badges
- Science topic