Fig. 1.
Fig. 1. RMCE. (A) Structure of a Lox Site: Lox sites are 34-bp double-stranded DNA fragments composed of two inverted binding sites for the CRE recombinase flanking an 8-bp spacer. The exact sequence of the spacer is not crucial for the recombination to occur, but the spacer sequence must be the same for two Lox sites to recombine.25 By introducing mutation in the spacer region, one can therefore create mutually incompatible Lox sites, that is, Lox sites that will recombine with themselves but not with each other. On the two last lines of the figure, the sequences of L1 and L2, the two mutually incompatible sites used in this study, are depicted. L1 and L2 differ by a G → A mutation at position +3 of their spacer regions. (B) Principle of RMCE: RMCE is a method that allows the exchange of a cassette located on a chromosome (cassette 1) by a cassette located on a plasmid (cassette 2). To perform RMCE a single-copy of cassette 1 flanked by L1 and L2 is placed on a chromosome by random integration (or by homologous recombination). A plasmid containing cassette 2 flanked by L1 and L2 is then cotransfected with a CRE expression plasmid in cells containing cassette 1. After the transfection, two reactions can occur (step 1): either the two L1 sites recombine or the two L2 sites recombine leading in either cases to integration of the plasmid carrying cassette 2 in the chromosome. If the L1 sites recombine leading in either cases to integration of the plasmid carrying cassette 2 in the chromosome. If the L1 sites recombine the structure on the left-hand side of the figure is obtained. If the L2 sites recombine the structure on the right-hand side of the figure is obtained. However, these structures are not the final outcome of the reaction because two L1 and L2 sites are now present in cis on the chromosome and can recombine at high efficiency (see text). These secondary recombinations (step 2) lead either to re-excision of the plasmid carrying cassette 2 or to the exchange of cassette 1 by cassette 2. When an insertion occur, it is stable because L1 and L2 cannot recombine with each other. Cassette 2 is therefore locked into the chromosome. The products of step 2 are also substrates for RMCE, the final proportion of exchanges versus re-excisions therefore depends on the number of copies of plasmid introduced in the cells. Theoretically, the equilibrium between exchange and re-excision can be tilted toward exchange by introducing large number of plasmid molecules in the cells. The thick lines represent chromosomal sequences. The double line plasmidic sequences. CAS1, cassette 1; CAS2, cassette 2. “X” symbolize CRE-mediated recombination.

RMCE. (A) Structure of a Lox Site: Lox sites are 34-bp double-stranded DNA fragments composed of two inverted binding sites for the CRE recombinase flanking an 8-bp spacer. The exact sequence of the spacer is not crucial for the recombination to occur, but the spacer sequence must be the same for two Lox sites to recombine.25 By introducing mutation in the spacer region, one can therefore create mutually incompatible Lox sites, that is, Lox sites that will recombine with themselves but not with each other. On the two last lines of the figure, the sequences of L1 and L2, the two mutually incompatible sites used in this study, are depicted. L1 and L2 differ by a G → A mutation at position +3 of their spacer regions. (B) Principle of RMCE: RMCE is a method that allows the exchange of a cassette located on a chromosome (cassette 1) by a cassette located on a plasmid (cassette 2). To perform RMCE a single-copy of cassette 1 flanked by L1 and L2 is placed on a chromosome by random integration (or by homologous recombination). A plasmid containing cassette 2 flanked by L1 and L2 is then cotransfected with a CRE expression plasmid in cells containing cassette 1. After the transfection, two reactions can occur (step 1): either the two L1 sites recombine or the two L2 sites recombine leading in either cases to integration of the plasmid carrying cassette 2 in the chromosome. If the L1 sites recombine leading in either cases to integration of the plasmid carrying cassette 2 in the chromosome. If the L1 sites recombine the structure on the left-hand side of the figure is obtained. If the L2 sites recombine the structure on the right-hand side of the figure is obtained. However, these structures are not the final outcome of the reaction because two L1 and L2 sites are now present in cis on the chromosome and can recombine at high efficiency (see text). These secondary recombinations (step 2) lead either to re-excision of the plasmid carrying cassette 2 or to the exchange of cassette 1 by cassette 2. When an insertion occur, it is stable because L1 and L2 cannot recombine with each other. Cassette 2 is therefore locked into the chromosome. The products of step 2 are also substrates for RMCE, the final proportion of exchanges versus re-excisions therefore depends on the number of copies of plasmid introduced in the cells. Theoretically, the equilibrium between exchange and re-excision can be tilted toward exchange by introducing large number of plasmid molecules in the cells. The thick lines represent chromosomal sequences. The double line plasmidic sequences. CAS1, cassette 1; CAS2, cassette 2. “X” symbolize CRE-mediated recombination.

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