In my experiment, I want to make a cell line expressing target gene + mCherry + luc .
(mCherry was selected because there is GFP in the cell line itself, and luciferase will be inserted for in vivo tracking.)
In this case, the target gene was inserted directly into the lentiviral plasmid (with mCherry + luc), not the cloning vector, and then transfection was performed with the package plasmid.
I've heard that you can use cloning vectors or use expression vectors directly.
If I have this purpose, please comment on how transfection should be done ...
Gene over-expression is the switching on of genes in aging cells. Gene over-expression in cultured cells is a common method used to examine the function of a gene. You could over-express in heterologous host or you use the combinatorial method. See these articles for details. Article Gene Overexpression: Uses, Mechanisms, and Interpretation
http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2373.html andA light-inducible CRISPR-Cas9 system for control of endogenous gene activation.pdf
You can clone and express in the same vector in certain cases. You can opt for shuttle vectors such as pAAV. Especially in viral transfection, any plasmid with a KOZAK sequence and relevant resistance should express seamlessly. Alternatively, you can try co-transfecting, or may be encode both the genes in the same vector, or you can even construct a fusion with the reporter gene.
Hi Jihyun,
I think that you could sub-clone your insert in a mamallian expression vector that already has everthing that you are looking for. You can look up pCDH-EF1a-eFFly-mCherry on Addgene to see if that would be suitable ( https://www.addgene.org/104833/)
Once that is done, you can transfect the vector into your cell of interest wihtout the need to produce lentiviruses.
Best,
Nicolas
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