New gene insertion in yeast - What are the suitable loci? (Feb/04/2008 )
I would like to introduce a new gene into S. cerevisiae. My question is where to insert it? I know I should be aware of the chromatin state in the target region. Has anyone done that already?
you can insert it about anywhere you want, provided you can select for insertion of your gene.
Asuming your favoured gene (yfg) is not a selectable marker or carries a selectable marker, it would be easier if you targetted the yfg to a proto-trophic gene such as LEU1 or URA3.
Best perhaps is URA3, as you can counterselect for uracil proto-trophy by FOA(5-fluroorotic acid). The yfg with targetting homology sequences comes along and replaces the URA3 gene by homologous recombination. The cell loses uracil proto-trophy and because a uracil auxotroph (need uracil to live). The FOA kills any cell with a functional ura3 gene. So in theory only cells which have lost the ura3 gene (hopefully meaning the yfg gene is now present). You can then test by PCR and southern to show targetting to the right place.
Alternatively, you don't care where the yfg gene is and care only that it is there. In that cause the yfg must be linked to a resistence marker. Throw the complex into the cell (maybe using either a fusion gene, self cleaving peptide, or ires for the marker) and select for the presence of the marker. Use PCR to show the construct is still intact. You will see difference in expression due to different location of targetting, but if you use a big mix of colonies that should even out the differences between cell types.
Thank you for your reply. My yeast strain is actually devoid of any marker genes. Plus, I would like to build a strategy that's independant of the strain I use. So I need to fuse my gene to a marker gene. But my question was are there standard regions that are used to integrate new genes beside that usual markers as you pointed (URA3, LEU2, etc) ?
I'm scared that if I choose a region at random, it will be a region in heterochromatin or a region with a special 2nd structure or a region important for regulation.
Thanks again and have a nice day!
To the best of my knowledge, unless you want to specifically knockout a gene, the proto-trophic gene (URA3, LEU2, ARG4, HIS3, ADE...etc) are the standard areas to target an exogenous gene too. Replacing these genes with the yfg is relatively easy as you can easily detect the lost of prototrophic gene. As mentioned, URA3 is favourite due to the FOA counter selection.
It is important to consider if you do want specific targetting. Screening for gene targetting can be quite labourous work and the experiment may not actually need specific targetting. Should random targetting be sufficient, it is common practice to compensate for any effects of where you gene landed by works on a pool of colonies (I work on a mix of 5-10) to obtain results.
As for linking your gene and marker....
If you knock out the URA3 gene, then you don't really need a marker.
You can link it by simply having two active genes with their own promoters sitting side by side. It simple but may have the danger of the two gene being unlinked during the targetting process. But you can screen by PCR to make sure both genes are still linked.
You can have a simple fusion gene. The danger of which is that the fusion protein may act funny for reason unknown.
You can have a fusion gene with self cleaving peptide-2A (scp).. yfg-scp-Marker.
Less likely for funny buisness.
Ah and best mentioned before I forget, you can have your gene floating around in an autonomous self replicating plasmid. As long as selection is maintained, the plasmid will remain.