Hi,
I have my gene of interest in pcDNA5/FRT/TO and would like to generate a stable cell line. Before generating the stable line, however, I would like to test expression by transient transfection.
Can the pcDNA5FRT/TO vector be used for transient expression? If so, do I need to add doxycycline to induce expression (as would be done with a stable line ...) or is the repressor only active when integrated into the genome?
In the handful of papers that I found, addition of doxycyclin during transient overexpression of a construct in pcDNA5/FRT/TO was not specifically mentioned.
Thank you,
Norbert
Hi Norbert,
You have the following options:
1. Transient transfection into a non-TRex cell line - TRex cell lines express the Tet repressor, so if you use any common cell line instead, you will have near-constitutive expression without adding doxycycline.
2. Transient transfection into a TRex cell line - You will need to add doxycycline to induce expression.
3. Stable transfection into a non-Flip-in cell line - transfection and antibiotic selection will produce a stable line.
4. Stable transfection into a Flip-in cell line - Transfection and antibiotic selection as above. However, this may be useful if you want your gene of interest to be integrated into the genome as a single copy as there is a single FRT recombination site in the Flip-in cell line. If the Flip-in cell line is also a TRex cell line, you will need to add doxycycline to induce gene expression, as in (2) above.
Sources:
http://tools.lifetechnologies.com/content/sfs/manuals/pcdna5frtto_man.pdf
http://www.lifetechnologies.com/order/catalog/product/R78007
Best,
Rao
Hi Norbert,
You have the following options:
1. Transient transfection into a non-TRex cell line - TRex cell lines express the Tet repressor, so if you use any common cell line instead, you will have near-constitutive expression without adding doxycycline.
2. Transient transfection into a TRex cell line - You will need to add doxycycline to induce expression.
3. Stable transfection into a non-Flip-in cell line - transfection and antibiotic selection will produce a stable line.
4. Stable transfection into a Flip-in cell line - Transfection and antibiotic selection as above. However, this may be useful if you want your gene of interest to be integrated into the genome as a single copy as there is a single FRT recombination site in the Flip-in cell line. If the Flip-in cell line is also a TRex cell line, you will need to add doxycycline to induce gene expression, as in (2) above.
Sources:
http://tools.lifetechnologies.com/content/sfs/manuals/pcdna5frtto_man.pdf
http://www.lifetechnologies.com/order/catalog/product/R78007
Best,
Rao
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