Single Vector multiple promoters? - (Sep/09/2008 )
Hi All,
I need to clone two different GPCRs into one vector and use it in transient transfections of HEK cells. In order to optimize transfection efficiency ( I am cloning into a stable cell line that is already stably expressing another GPCR) I would like to keep everything in one vector. It appears the pIRES vectors may be unsuitable as I think you cannot put a stop codon after the first upstream which means adding extra AAs to the upstream receptor insert and my downstream insert would be too large (2.5kB) for efficient expression through the IRES.
Does anyone know of a good vector with two MCS sites that are under the control of independent promoters that can be cloned into E. Coli and expressed in mammalian cells that may be suitable for what I am trying to do?
Thanks for the help
We have made these type of vectors ourselves. two independent cassettes in one plasmid. These are different viral vectors.
Hi,
I was going to make these type of vector too. which promoters did you put together?
Thanks for the reply. Do you have any info or citations on how these work and how to make them?
Hi,
I was going to make these type of vector too. which promoters did you put together?
I have used human synapsin as both promoters for one vector and for other murine CMV and human synapsin.
There is this other where human CMV was used for both cassettes.
Check this paper from my old lab. [attachment=5277:paper1.pdf]
Thanks for the info- very cool
Do you know if any plasmid vectors exist that use multiple cassettes without using an IRES or if this is even possible?
There is this other where human CMV was used for both cassettes.
Check this paper from my old lab. [attachment=5277:paper1.pdf]
I think having more than 2 casettes is possible. As the vectors I described ofcourse have an ampicillin resistance gene and its promoter. SO basically these have 3 casettes.
Does this answer your question?
Do you know if any plasmid vectors exist that use multiple cassettes without using an IRES or if this is even possible?
Yes, it is most certainly possible. You can even build your own shuttle vector that expresses several genes under different promoters for say S.pombe, caries an 2 S.pombe markers and a E coli marker for selection in e coli.
The only problem you can experience is if you use the same promoter or terminator two or more times. Having repeated structures in your plasmid can cause some problems due to homologous recombination. (especially when working with E coli and yeast)
Thanks for help, much appreciated
Do you know if any plasmid vectors exist that use multiple cassettes without using an IRES or if this is even possible?
Yes, it is most certainly possible. You can even build your own shuttle vector that expresses several genes under different promoters for say S.pombe, caries an 2 S.pombe markers and a E coli marker for selection in e coli.
The only problem you can experience is if you use the same promoter or terminator two or more times. Having repeated structures in your plasmid can cause some problems due to homologous recombination. (especially when working with E coli and yeast)