Team:USP-UNESP-Brazil/Plasmid Plug n Play/Background

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Concerning the molecular mechanism of recombination, a single recombinase molecule binds to each palindromic half of a loxP site, then the recombinase molecules form a tetramer, thus bringing two loxP sites together (8). The recombination occurs within the spacer area of the loxP sites. The post-recombination loxP sites are formed from the two complementary halves of the pre-recombination sites (Fig. 1). This mechanism was further improved for science research purposes, and nowadays, lots of tools have been made based on it. The best-known example is the Nobel Prize winner experiment of knockout on rats using the Cre-lox recombination (4).
Concerning the molecular mechanism of recombination, a single recombinase molecule binds to each palindromic half of a loxP site, then the recombinase molecules form a tetramer, thus bringing two loxP sites together (8). The recombination occurs within the spacer area of the loxP sites. The post-recombination loxP sites are formed from the two complementary halves of the pre-recombination sites (Fig. 1). This mechanism was further improved for science research purposes, and nowadays, lots of tools have been made based on it. The best-known example is the Nobel Prize winner experiment of knockout on rats using the Cre-lox recombination (4).
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=figplugplay.jpg | caption=FIG. 1. Cre/loxP system. (A) Close-up of Cre recombinase-mediated recombination between two 34 bp loxP sites. Schematics of excision vs. integration (B) and inversion (C) (5).  | size=200px }}
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{{:Team:USP-UNESP-Brazil/Templates/RImage | image=figplugplay.jpg | caption=FIG. 1. Cre/loxP system. (A) Close-up of Cre recombinase-mediated recombination between two 34 bp loxP sites. Schematics of excision vs. integration (B) and inversion (C) (5).  | size=500px }}
Now we will talk about DNA integration or insertion. It is another way of working using Cre-lox recombination between two different DNA fragments. It has already been extensively explored and is the key mechanism of our experiment. Using two DNA fragments, each one with one loxP site, is possible to fuse both into one larger fragment.  
Now we will talk about DNA integration or insertion. It is another way of working using Cre-lox recombination between two different DNA fragments. It has already been extensively explored and is the key mechanism of our experiment. Using two DNA fragments, each one with one loxP site, is possible to fuse both into one larger fragment.  
However, insertion of a circular DNA carrying a loxP into a loxP site on a chromosome (integrative recombination) is quite inefficient because unimolecular reactions are kinetically favored over bimolecular reactions, causing that the inserted DNA will often be excised (6) (Fig. 2a). To tackle this problem we have used loxP mutant sites (2). These sites help to maintain the inserted DNA in the chromosome (Fig. 2b).In our project we used one loxP site and two loxP mutant sites (lox71 and lox66) (Fig. 2c).
However, insertion of a circular DNA carrying a loxP into a loxP site on a chromosome (integrative recombination) is quite inefficient because unimolecular reactions are kinetically favored over bimolecular reactions, causing that the inserted DNA will often be excised (6) (Fig. 2a). To tackle this problem we have used loxP mutant sites (2). These sites help to maintain the inserted DNA in the chromosome (Fig. 2b).In our project we used one loxP site and two loxP mutant sites (lox71 and lox66) (Fig. 2c).

Revision as of 15:06, 21 September 2012