Multiplexing RMCE: versatile extensions of the Flp-recombinase-mediated cassette-exchange technology.
There are strong indications, but as yet no proof, that extended 48-bp Flp recombinase targets (FRTs) represent unique targets in all eukaryotic genomes investigated, and that recombinase-mediated cassette exchange is not hampered by the occurrence of genomic pseudo sites. This encouraged the present study in which we explore the feasibility of exchanging, in a given cell, two distinct genomically anchored cassettes, each flanked by a unique set of two heterospecific FRT sites. Mutant FRTs have to meet two major prerequisites for successful recombinase-mediated cassette exchange: (i) a self-recognition capacity comparable to a pair of FRT wild-type sites (FRTxFRT), and (ii) a negligible cross-interaction if part of a set of heterospecific sites (F'xF). We apply a two-step strategy to explore various newly created FRT spacer mutants for these properties. As a result of our screening steps, we identify combinations of sites that are successfully applied to parallel Flp-mediated genomic targeting ("multiplexing") reactions (i.e., the simultaneous exchange of two separate target cassettes in a given cell).
Turan
Soeren
S
Hannover Medical School OE6960 Experimental Hematology, Carl-Neuberg-Strasse 1, D-30625 Hannover,Germany.
Kuehle
Johannes
J
Schambach
Axel
A
Baum
Christopher
C
Bode
Juergen
J
D004254:DNA Nucleotidyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting; D006801:Humans; D009154:Mutation / Q000235:genetics; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000235:genetics*; D011995:Recombination, Genetic; D013937:Thymidine Kinase / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C063242:hygromycin-B kinase; D013937:Thymidine Kinase; D004254:DNA Nucleotidyltransferases; C045119:FLP recombinase