microhomologous recombination

Marc Le Bert lebert at cnrs-orleans.fr
Thu Mar 15 18:45:56 EST 2001


Hi everybody,



I have something like a brainstorm about the use of yeast to construct with ease and speed the targeting vectors we use to knockout genes in mice.


My first question is about microhomologous recombination (60pb) in yeast to modify YACs or adapted  BACs or PACs to introduce modifications. Is it so easy and sure? (I never use yeast, generally we make a complex targeting vector at best in 3 to 6 months!) Are there any specific strains? Which protocol do we use?


My second question is about the promoters. Are there dual promoters that can work both in yeast and mammalian cells? Generally I use PGK, HSVTK promoters.


My third question is about selection genes. What are the best selection cassettes to work with? I want to shuttle my modified YACs or BACs from yeast to mammalian cells with the same selection (G418, puromycine or hygromycine). If not, I have to use both selection in my cassette...may be its too large to be convenient! I want also to use the flp and FRT mutants sites to introduce sequential modifications in my targeting vector.


And the last question is about the availability of vectors, strains, cassettes...can someone help me to find the holy yeastland


Thanks for giving me your thoughts

Regards,

Marc




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