PMC:1540739 / 3416-5012 JSONTXT

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For example, exchanges using cassettes with directly repeated loxP sites were inefficient because excisions dominated the intended exchanges (8). loxP sites with different sequences were generated to overcome this problem, but these sites also underwent intramolecular recombination, making RMCE efficient only if the replacement cassette contained a marker enabling selection of the desired recombinant (7,9–12). However, interference resulting from expression of the selection marker and the endogenous gene (13) necessitates strategies to remove the selectable gene. Together, previous studies indicate that an optimal RMCE requires (i) inverted heterologous loxP sites diverging by at least 2 nt to maximize the efficiency of exchange and (ii) an expression cassette enabling both positive selection to identify the initial recombinant and then negative selection to obtain a ‘marker-free’ mutant allele 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