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Mouse Transgenesis - Generating DNA for microinjection


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#1 BSM

BSM

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Posted 10 August 2011 - 09:35 PM

Hey everyone,

I have not performed any molecular cloning in years and was looking for some advice. I have recently acquired transgenic mice that express the reverse tetracycline-controlled transactivator protein under the control of a cell specific promoter. In the presence of doxycycline, cells that express the transactivator protein can bind to a tetracycline-responsive promoter element(tetO) and drive expression of a gene of interest. It is my goal to generate a second transgenic mouse line that contains the tetO operator fused to a minimal CMV promoter, my gene of interest an IRES sequence and then a fluorescent marker like red fluorescent protein. I know that there are vectors out there that contain the TetO operator and other that contain the IRES sequences for single mRNA expression but protein expression of the gene of interest and the fluorescent marker. Where should I start here? Can the tetO operator be used with other highly active promoters? My goal is to amplify this plasmid, digest out and purify my transgene then microinject into zygotes.

Thanks for the help.

Edit// It looks like clontech's 2nd generation Tet-On systems may be what I am looking for. Anyone have experience with these? I noticed that according to this map: http://www.clontech....p?cItemId=17966

there are no restriction sites after the polyA sequences which means I would not be able to cut the plasmid at both PolyA ends. Bummer

Edited by BSM, 10 August 2011 - 10:32 PM.





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