After amplifying a promoter sequence, transgene and activation domain and polyA signal. I plan to insert these constructs into a pUC19 vector with a downstream promoter CMV associated with GFP and express the plasmid in mammalian cells invivo. Would there be expression (there should be) or should I choose a different vector bicistronic vector? The promoter is tissue specific thus can drive gene expression.
Hi there,
pUC19 is a bacterial vector made for cloning/expression in bacteria. It is naturally devoided of any selection marker for eukaryotic cell transfection.
It should work - similar to any other backbone (if you want to do just a transient transfection).
When investigating different promoter elements it may be beneficial to use a backbone of pGL4 (Promega) since they have removed a lot of potential transcription factor binding sites from the backbone (that sometimes may cause spurious expression). Reference: Researchgate answer from Promega (https://www.researchgate.net/post/Problem_with_luciferase_reporter_assay) and pGL4 manual (https://www.promega.com/resources/protocols/technical-manuals/0/pgl4-luciferase-reporter-vectors-protocol/).
As a rule of thumb, try to avoid repeating sequences: if you use 2 promoters or 2 poly(A) sequences, choose different ones .
Alternatively you can use an IRES or a 2A peptide between your transgene and the GFP but only if they are in the same direction.
Also, best to add a Kozak sequence after the promoter...
Your description is not clear. pGL is a luciferase vector . Where are you going to insert your transgene and the CMV-GFP? In any case this is not the best vector choice. Other plasmids are more convenient to insert and express a transgene fused, not fused or IRES-ed to GFP depending on your choice or purpose.
GFP would be cleaved from GFP containing vector. pGL is just an example to describe the components an expression vector has. Both genes would be inserted into MCS in the same direction however they need be interrupted in between so that they don't end up as a fusion protein. Aim is to just express.
if you have the construct made is worth testing immediately rather than theorizing if it is going to work or not.
hopfully working i currently working on the same direction have you
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