I am working on a protein whose loss is lethal for cell viability. Hence, we are looking for an inducible lentiviral construct to deplete the endogenous protein pool and at the same time, we want to reintroduce the epitope tagged version of the same protein (mutant form) to study its functions.
Does anyone has tried to make this kind of construct? Or anyone knows whether this type of vector is commercially available?
hi Varun,
I had a positive experience for my purposes with lentiviral silencing vector from Thermo: https://www.thermofisher.com/ru/ru/home/life-science/dna-rna-purification-analysis/napamisc/vector-maps/psilencer-5-1-u6-retro-vector-map.html
In general, I would like to say YES. However, you have to design an expression cassette including promoter ( inducible), terminator and other elements especially internal ribosome entry site (IRES). You can if you have two open reading frames (ORF) do not matter coding or non coding you can choose a suitable IRES for your cassette. Please be aware that you have to clone your non coding gene upstream of your coding gene. Then you can design a pair of primers and subclone your cassette to any desired expression vector. Notable, you have to break the promoter of expression vector because you do not need it anymore. In fact you have got your desired promoter but steal need to selection marker! It is the main reason that you need to subclone your cassette into the expression vector. If all above are confusing, find a ready to use viral vector and just subclone your coding or non coding gene in a right place!
Hi Varun,
You can use L309 lentiviral construct which has U6 and H1 promoter, and EGFP. You can insert the shRNA sequence and over expressing shRNA resistant sequence in the same construct. Hope it works for you.
- Badges
- Science method
- Similar topics
- Biological Science
- Genetics
- Gene Expression
- Protein Expression