I want to deplete a gene X in yeast using the TetOff and AID system at the same time. I transformed two strains in TetO-X and X-AID and I am now asked to do backcross to get TetO-X-AID.
So, I am wondering, why should I do tetrad dissections and cross my finger for a recombination to happen inside my gene when I could certainly get my final strain with just a second transformation.
That's what I was initially thinking but I was asked to proceed with backcross and not transformation. I know I have very low probabilities to get my construct, but I hope there is a reason to this protocol. I imagine there must be some pros at using backcross, like a cleaner sequence because of recombination or something like this.
Hi Terry,
First of all, Alexander is absolutely right. Depending on how long your gene is, the probability that you get recombination within your genes ORF is almost zero. Be aware though, that you need the skp-TIR1 fusion gene in you genetic background for the AID to work. Maybe that's whats meant here?
What I would do is (for essential proteins): Make a plasmid with tetO-X-AID and antibiotic marker e. g. kanMX/G418. Delete your gene X in a diploid ade6-M210/ade6-M216 strain with a second antibiotic marker, e. g. hphMX/Hygromycin. Introduce your tetO-X-AID-hphMX construct in this strain and tetrad dissect. In case the fusion protein is not functional you should see that the tetO-X-AID does not complement the deletion. Then you should figure out if its the promoter or the tag. If it does complement the deletion, get a h+, ade6-, hphMX+, kanMX clone from your tetrad dissection and cross it to the skp1-TIR1 strain (the marker of this strain is ade6, if you are using the one from Kanke et al). This is as clean as it gets from my point of view. Quite a bit of work though.
If you want less work delete your endogenous gene with the tetO-X-AID construct directly in a diploid background.
Hope that helped?
Hello again everyone.
First, Mr. Lorenz, I have resistance markers on both sides. Construction is like this : Hyg-TDH3pro-TetO-X-AID-TEFpro-Kan. For more details, I am using the iAID system from S. Tanaka (fig.2) http://www.readcube.com/articles/10.1002%2Fyea.3080
Second, Christoph, my construct is endogenous so I am directly transforming with PCR product modified to get some homologous parts with my gene. As for my Tir1, It should be expressed by plasmid, so I don't think that's the reason.
Thank you all for your responses and your attention !
P.S : I finally got to talk with my professor and we agreed that it was not the best way to go about it. Finally, he orientated me on random spore selection, which is, in my opinion, more interesting. I am still open to tips since I have never done this before.
Thank you very much. I will keep this in mind in case the random spore method do not work.
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