respectively. Base Substitutions in the capitalized nucleotides led to the greatest reduction in FLP-mediated site-specific recombination (Wildtype x mutant and mutant x mutant): 5'3' Many available constructs include an additional arm sequences (5'--3') one base pair away from the upstream element and in the same orientation: 5'3' This segment is dispensable for excision but essential for integration, including Recombinase-mediated cassette exchange. Because the recombination activity can be targeted to a selected organ, or a low level of recombination activity can be used to consistently alter the DNA of only a subset of cells, Flp-FRT can be used to construct genetic mosaics in multicellular organisms. Using this technology, the loss or alteration of a gene can be studied in a given target organ of interest, even in cases where experimental animals would not survive the loss of this gene in other organs (spatial control). The effect of altering a gene can also be studied over time, by using an inducible promoter to trigger the recombination activity late in development (temporal control) - this prevents the alteration. Biochemical structure of Flp and mechanism of action Biochemically relevant structure and active site The Flp protein, much like Cre, is a tyrosine family site-specific recombinase. This family of recombinases performs its function via a type IB topoisomerase mechanism causing the recombination of two separate strands of DNA. Recombination is carried out by a repeated two-step process. The initial step causes the creation of a Holliday junction intermediate. The second step promotes the resulting recombination of the two complementary strands. As their family name suggests, a highly conserved tyrosine nucleophile cleaves the DNA strands. The nucleophilic properties of the tyrosine attack and bind to the 3'-phosphate at the point of DNA cleavage. The resulting 5'-hydroxyl group of the cleaved DNA acts as the nucleophile and attacks the 3'-phosphate on the complementarily cleaved DNA strand, resulting in successful recombination. Outside of the tyrosine residue, there is a conserved catalytic pentad. This pentad is made up of a lysine (Lysβ), two arginines (Arg I and II), a histidine (His-II), and a histidine/tryptophan (His/Trp-III) that comprises a mandatory and highly conserved constellation of residues for the active site of Flp and Cre (along with other IB topiosomerases). These other residues are crucial to the correct orientation of Flp binding and positioning on the DNA strands. Application of FLP-mediated site-specific recombination Initial problems Thermolability Initial application of the FLP-FRT