Hello, I would like to remove selection cassette from my mutant plant in order to transform another construct with same selection. I know, cre/lox recombination is one of the possibility, but couldn't find the detailed protocol for the same.
Can someone please share with me the protocol or tell me where to look for.
Thanks.
Hi Anamika,
This is usually how people perform (of course, there are some more complicated strategies):
1. Everything you place in-tween the two Cre recombinase-recognize recombination sites (called 'lox') will be looped out (excised) through Cre-mediated site-specific recombination.
2. If you want to remove the SMG (selectable marker gene) later with Cre-lox system, the only extra thing you have to do during building your regular transformation vector is adding 2 lox sites to flank the SMG: -lox-SMG-lox-
3. So, your transgenic lines (name 'A') will have gene-of-interest (GOI) and -lox-SMG-lox-
4. You also need to develop a Cre line to provide Cre-recombinase; let's call them 'B' lines**
5. Finally, you make cross (line A x line B) to remove SMG, upon the expression of Cre gene
** for the Cre lines, you need to screen out a few single-copy lines, and use them to cross the target lines (which with -lox-SMG-lox-). We need 'a few' lines because due to 'position effect', some lines will express well and some don't)
I attached a figure (from web), so you can better understand. The 'black' triangle represent lox site. Everything flanked by the lox sites will be excised out as a 'circular molecule'.
Hi Yuan-Yeu Yau, thanks for the info. Actually I work with mosses, and for some reason I am never successful in crossing the moss lines.
I have mutant moss lines, and a cre-plasmid, and was told to transform this plasmid to mutants; but have no detailed info. of the protocol!
One more thing, if I have two selection cassettes (both flanked by lox sites), then can I get rid of both at the same time?
Hi Anamika,
1. You need to know why you are transforming the mutant line:
(1) Are you just want to create a Cre-expressing line?
(2) Does the mutant line already have a lox-SMG-lox cassette in its genome? If so, you can re-transform the mutant with Cre plasmid to remove the SMG. You don't need a specific protocol to transform the mutant with your Cre plasmid. Just transform it as the way you transform a regular plasmid. Once you get a positive transformant (now you have both lox-SMG-lox and Cre-gene cassette), you can perform PCR to see whether the SMG has been removed. And move to next generation.
So, check out with your advisor what is in the mutant line.
2. Regarding your second question: yes, theoretically, you can remove both fragments, as long as they are flanked with lox sites.
- Badges
- Science topic
- Similar topics
- Biological Science
- Botany
- Plant Biology
- Plant Biotechnology