218:
conditional mouse alleles carrying single or multiple loxP and FRT sites. dual RMCE (dRMCE; Osterwalder et al., 2010) was recently developed as a re-engineering tool applicable to the vast numbers of mouse conditional alleles that harbor wild-type loxP and FRT sites and therefore are not compatible with conventional RMCE. The general dRMCE strategy takes advantage of the fact that most conditional alleles encode a selection cassette flanked by FRT sites, in addition to loxP sites that flank functionally relevant exons ('floxed' exons). The FRT-flanked selection cassette is in general placed outside the loxP-flanked region, which renders these alleles directly compatible with dRMCE. Simultaneous expression of Cre and Flp recombinases induces cis recombination and formation of the deleted allele, which then serves as a 'docking site' at which to insert the replacement vector by trans recombination. The correctly replaced locus would encode the custom modification and a different drug-selection cassette flanked by single loxP and FRT sites. dRMCE therefore appears as a very efficient tool for targeted re-engineering of thousands of mouse alleles produced by the IKMC consortium.
227:
72:
86:-site (F). If a gene cassette is flanked by a set of these sites (F and Fn, for example) it can change places, by double-reciprocal recombination, with a second cassette that is part of an exchange plasmid (Figure 1, part A). A model experiment is shown in part C, in which an ´empty´ cell is modified by either a standard
173:(cross-hatched half-arrows in Figure 1). Each mutant Fn recombines with an identical mutant Fn with an efficiency equal to the wildtype sites (F x F). A cross-interaction (F x Fn) is strictly prevented by the particular design of these components. This sets the stage for the situation depicted in Figure 1A:
59:. As a consequence the newly introduced information may not be realized (expressed), the gene(s) may be lost and/or re-insert and they may render the target cells in unstable state. It is exactly this point where RMCE enters the field. The procedure was introduced in 1994 and it uses the tools yeasts and
237:
sites (F5/F3-F/Fn) is introduced into the genome. The unique F5/F3 address can then be used to introduce an upstream-regulatory element and the F/Fn address to apply a similar modification at the downstream end. after the expression of the gfp-reporter has been optimized by systematic changes of this
303:
s corresponds to their initial state. This property enables the intentional, repeated mobilization of a target cassette by the addition of a new donor plasmid with compatible architecture. These "multiplexing-RMCE" options open unlimited possibilities for serial- and parallel specific modifications
246:
site and a mutant called "n") or each Fn-Fm pair (consisting of two mutants, "m" and "n") constitutes a unique "address" in the genome. A prerequisite are differences in four out of the eight spacer positions (see Figure 1B). If the difference is below this threshold, some cross-interaction between
181:
the GOI (gene-of-interest) is part of a circular ´exchange plasmid´ and is flanked by a set of matching sites. This exchange plasmid can be introduced into the cell at large molecular excess and will thereby undergo the depicted exchange (RMCE-) reaction with the pre-selected genomic address (i.e.
217:
It has been previously established that coexpression of both Cre and Flp recombinases catalyzes the exchange of sequences flanked by single loxP and FRT sites integrated into the genome at a random location. However, these studies did not explore whether such an approach could be used to modify
258:. These modifications can be driven to completion in case the compatible donor plasmid(s) are provided at an excess (mass-action principles). Figure 2 illustrates one use of the multiplexing principle: the stepwise extension of a coding region in which a basic expression unit is provided with
90:
approach or by RMCE. Please note that in the first case multiple genomic sites are hit, each giving raise to a different expression level (cf. the broad distribution of green dots). If a pre-defined genomic address is used to introduce the same gene reporter, each
177:
a target cassette (here a composite +/- selection marker) is flanked by an F- and an Fn site. After its introduction into the genome of a host cell the properties of many integration sites (genomic ´addresses´) are characterized and appropriate clones are
914:
Turan, Soeren; Galla, Melanie; Ernst, Ellen; Qiao, Junhua; Voelkel, Christine; Schiedlmeier, Bernhard; Zehe, Christoph; Bode, Juergen (2011). "Recombinase-Mediated
Cassette Exchange (RMCE): Traditional Concepts and Current Challenges".
254:(for instance F-Fn and Fm-Fo). These addresses will be recognized by donor plasmids that have been designed according to the same principles, permitting successive (but also synchronous) modifications at the predetermined
238:
type, the central reporter cassette can be exchanged for any ´gene-of-interest´(GOI): the GOI will be flanked by the F3 and F-sites, respectively and introduced accordingly while the flanking elements will remain in place
38:
The genetic modification of mammalian cells is a standard procedure for the production of correctly modified proteins with pharmaceutical relevance. To be successful, the transfer and expression of the
641:"Mutant Lrp1 knock-in mice generated by recombinase-mediated cassette exchange reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development"
493:
Bode, J; T. Schlake; M. Iber; D. SchĂĽbeler; J. Seibler; E. Snezhkov; L. Nikolaev (2000). "The transgeneticist's toolbox - Novel methods for the targeted modification of eukaryotic genomes".
205:
Flp, this novel procedure is not only relevant to the rational construction of biotechnologically significant cell lines, but it also finds increasing use for the systematic generation of
875:
Qiao, J.; Oumard, A.; Wegloehner, W.; Bode, J. (2009). "Novel Tag-and-Exchange (RMCE) Strategies
Generate Master Cell Clones with Predictable and Stable Transgene Expression Properties".
604:
Cesari F, Rennekampff V, Vintersten K, Vuong LG, Seibler J, Bode J, Wiebel FF, Nordheim A (Feb 2004). "Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange".
725:
Cesari F, Rennekampff V, Vintersten K, Vuong LG, Seibler J, Bode J, Wiebel FF, Nordheim A (2004). "Elk-1 knock-out mice engineered by Flp recombinase-mediated cassette exchange".
281:
the first RMCE-based modification has occurred. This is due to the fact that each phiC31-catalyzed exchange destroys the attP and attB sites it has addressed converting them to
325:
Insertion of a target cassette in a mammalian host cell line (CHO DG44 in suspension culture) and exchange with an ER stress reporter construct via targeted integration (RMCE).
292:
product sites, respectively. While these changes permit the subsequent mounting of new (and most likely remote) targets, they do not enable addressing several RMCE targets
944:
Turan, S; Zehe, C; Kuehle, J; Qiao, J; Bode, J (2013). "Recombinase-Mediated
Cassette Exchange (RMCE) – a rapidly-expanding toolbox for targeted genomic modifications".
528:
Turan, S.; Kuehle, J.; Schambach, A.; Baum, C.; Bode, J. (2010). "Multiplexing RMCE: Versatile
Extensions of the Flp-Recombinase-Mediated Cassette-Exchange Technology".
721:
J. Bode, S. Götze, M. Klar, K. Maaß, K. Nehlsen, A. Oumard & S. Winkelmann (2004) BIOForum 34-36 Den Viren nachempfunden: Effiziente
Modifikation von Säugerzellen.
427:
756:"Mutant Lrp1 knock-in mice generated by RMCE reveal differential importance of the NPXY motifs in the intracellular domain of LRP1 for normal fetal development"
99:
Most yeast strains contain circular, plasmid-like DNAs called "two-micron circles". The persistence of these entities is granted by a recombinase called
688:
Kober L, Zehe C, Bode J (October 2012). "Development of a novel ER stress based selection system for the isolation of highly productive clones".
47:
are based on the same principles. Traditional procedures used for transfer of GOIs are not sufficiently reliable, mostly because the relevant
334:
282:
274:
202:
166:
116:
71:
226:
27:
allowing the systematic, repeated modification of higher eukaryotic genomes by targeted integration, based on the features of
209:. Stem cells can be used to replace damaged tissue or to generate transgenic animals with largely pre-determined properties.
975:"Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases"
1085:
189:
is a process that can be repeated with the same or a different exchange plasmid ("serial RMCE"). Please note that RMCE
1090:
339:
28:
247:
the mutants may occur leading to a faulty deletion of the sequence between the heterospecific (Fm/Fn or F/Fn) sites.
250:
13 FRT-mutants have meanwhile become available, which permit the establishment of several unique genomic addresses
834:"Recommended Method for Chromosome Exploitation: RMCE-based Cassette-Exchange Systems in Animal Cell Biotechnology"
1080:
1016:
Osterwalder, Marco; Galli, Antonella; Rosen, Barry; Skarnes, William C; Zeller, Rolf; Lopez-Rios, Javier (2010).
51:
influences have not been sufficiently explored: transgenes integrate into chromosomes with low efficiency and at
364:
43:
has to be highly efficient and should have a largely predictable outcome. Current developments in the field of
799:"Talking about a revolution: the impact of site-specific recombinases on genetic analyses in mice. Develop"
421:
359:
354:
344:
142:
125:
112:
100:
296:, nor do they permit "serial RMCE", i.e. successive, stepwise modifications at a given genomic locus.
259:
263:
138:
565:"Site-specific recombinases: from tag-and-target- to tag-and-exchange-based genomic modifications"
1065:
165:
This spectrum of options could be extended significantly by the generation of spacer mutants for
754:
Roebroek, A. J. M.; Reekmans, S.; Lauwers, A.; Feyaerts, N.; Smeijers, L.; Hartmann, D. (2006).
1047:
1004:
961:
932:
902:
863:
820:
785:
742:
705:
670:
621:
586:
545:
510:
475:
409:
233:. In the given example a reporter gene cassette (gfp/tk/neo), flanked by four heterospecific
1037:
1029:
994:
986:
953:
924:
892:
884:
853:
845:
810:
775:
767:
734:
697:
660:
652:
613:
576:
537:
502:
465:
457:
446:"Site-Specific Transformation of Drosophila via phiC31-Integrase-Mediated Cassette Exchange"
401:
92:
24:
349:
56:
197:(dotted lines) that would otherwise trigger immunologic or epigenetic defense mechanisms.
1042:
1017:
858:
833:
780:
755:
665:
640:
470:
445:
60:
999:
974:
815:
798:
1074:
32:
317:
Generation of transgenic knock-out/-in mice and their genetic modification by RMCE.
31:
processes (SSRs). For RMCE, this is achieved by the clean exchange of a preexisting
639:
Roebroek AJ, Reekmans S, Lauwers A, Feyaerts N, Smeijers L, Hartmann D (Jan 2006).
242:
Multiplexing setups rely on the fact that each F-Fn pair (consisting of a wildtype
87:
44:
771:
656:
461:
79:
48:
957:
107:
of this enzyme associate with two identical short (48 bp) target sites, called
82:(´Flp´) from yeast. Part B shows mutants (Fn) of the naturally occurring 48 bp
928:
888:
849:
541:
206:
63:
have evolved for the efficient replication of important genetic information:
40:
1051:
1008:
965:
936:
906:
867:
824:
789:
746:
709:
674:
625:
590:
549:
514:
479:
413:
990:
581:
564:
104:
506:
405:
388:-)sites for the exchange of expression cassettes at defined chromosomal
277:, which permits introduction of another RMCE target at a secondary site
1033:
897:
738:
617:
701:
384:
Schlake, T., Bode, J. (1994). "Use of mutated Flp-recognition-target-(
129:
of a sequence that is flanked by two identical but inversely oriented
95:
derived from such an event shows comparable expression characteristics
299:
This is different for Flp-RMCE, for which the post-RMCE status of
225:
70:
321:
Tagging and cassette exchange in DG44 cells in suspension culture
153:
the inefficient reversion of the letter process, commonly called
35:
for an analogous cassette carrying the "gene of interest" (GOI).
1018:"Dual RMCE for efficient re-engineering of mouse mutant alleles"
146:
of a sequence that is flanked by two equally oriented identical
1066:
https://www.sciencedaily.com/releases/2011/11/111130115822.htm
273:
A recent variation of the general concept is based on PhiC31
973:
Lauth, M.; Spreafico, F.; Dethleffsen, K.; Meyer, M (2002).
832:
Oumard, A.; Qiao, J.; Jostock, T.; Li, J.; Bode, J. (2006).
78:
exchange of genetic cassettes (´flip´ step) is enabled by a
157:
or "addition" of an extra piece of DNA carrying a single
193:
of the GOI at the pre-determined locus and that it does
444:
Bateman, Jack R; Anne M. Lee; C.-ting Wu (June 2006).
55:that provide only sub-optimal conditions for their
111:("flip-recombinase targets"), resulting in their
115:. The outcome of such a process depends on the
195:not co-introduce prokaryotic vector sequences
8:
426:: CS1 maint: multiple names: authors list (
1041:
998:
896:
857:
814:
779:
664:
580:
469:
376:
419:
21:recombinase-mediated cassette exchange
439:
437:
119:of the participating FRTs leading to
7:
797:Branda, C.S.; Dymecki, S.M. (2004).
335:Site-specific recombinase technology
14:
161:site identical to the target site
313:Generation of transgenic animals
304:of pre-determined RMCE-targets
275:(an integrase of the Ser-class)
1:
816:10.1016/S1534-5807(03)00399-X
772:10.1128/MCB.26.2.605-616.2006
657:10.1128/MCB.26.2.605-616.2006
917:Journal of Molecular Biology
182:the F <+/-> Fn target)
76:Figure 1: Principle of RMCE:
462:10.1534/genetics.106.056945
340:Site-specific recombination
231:Figure 2: Multiplexing RMCE
29:site-specific recombination
1107:
958:10.1016/j.gene.2012.11.016
563:Turan, S; J. Bode (2011).
929:10.1016/j.jmb.2011.01.004
889:10.1016/j.jmb.2009.05.012
850:10.1007/s10616-006-6550-0
542:10.1016/j.jmb.2010.07.015
365:Homologous recombination
191:introduces just one copy
239:
201:First applied for the
96:
360:Genetic recombination
355:Cre-Lox recombination
345:FLP-FRT recombination
229:
74:
582:10.1096/fj.11-186940
117:relative orientation
23:) is a procedure in
1086:Genetics techniques
507:10.1515/BC.2000.103
406:10.1021/bi00209a003
400:(43): 12746–12751.
101:"flippase" or "Flp"
1091:Molecular genetics
1034:10.1038/nmeth.1521
991:10.1093/nar/gnf114
803:Developmental Cell
739:10.1002/gene.20003
690:Biotechnol. Bioeng
618:10.1002/gene.20003
270:-acting elements.
260:genomic insulators
240:
97:
67:General principles
979:Nucleic Acids Res
702:10.1002/bit.24527
575:(12): 4088–4107.
501:(9–10): 801–813.
222:Multiplexing RMCE
1098:
1081:Applied genetics
1055:
1045:
1012:
1002:
969:
940:
910:
900:
871:
861:
828:
818:
793:
783:
750:
714:
713:
696:(10): 2599–611.
685:
679:
678:
668:
636:
630:
629:
601:
595:
594:
584:
560:
554:
553:
525:
519:
518:
490:
484:
483:
473:
441:
432:
431:
425:
417:
381:
25:reverse genetics
1106:
1105:
1101:
1100:
1099:
1097:
1096:
1095:
1071:
1070:
1062:
1028:(11): 893–895.
1015:
972:
943:
913:
874:
844:(1–3): 93–108.
831:
796:
760:Mol. Cell. Biol
753:
724:
718:
717:
687:
686:
682:
638:
637:
633:
603:
602:
598:
562:
561:
557:
527:
526:
522:
492:
491:
487:
443:
442:
435:
418:
383:
382:
378:
373:
350:Cre recombinase
331:
323:
315:
310:
224:
215:
203:Tyr-recombinase
167:extended 48 bp
69:
12:
11:
5:
1104:
1102:
1094:
1093:
1088:
1083:
1073:
1072:
1069:
1068:
1061:
1060:External links
1058:
1057:
1056:
1022:Nature Methods
1013:
970:
941:
923:(2): 193–221.
911:
883:(4): 579–594.
872:
838:Cytotechnology
829:
794:
766:(2): 605–616.
751:
722:
716:
715:
680:
631:
596:
555:
520:
485:
456:(2): 769–777.
433:
375:
374:
372:
369:
368:
367:
362:
357:
352:
347:
342:
337:
330:
327:
322:
319:
314:
311:
309:
306:
295:
280:
253:
223:
220:
214:
211:
199:
198:
196:
192:
187:RMCE-principle
183:
179:
163:
162:
151:
134:
68:
65:
61:bacteriophages
13:
10:
9:
6:
4:
3:
2:
1103:
1092:
1089:
1087:
1084:
1082:
1079:
1078:
1076:
1067:
1064:
1063:
1059:
1053:
1049:
1044:
1039:
1035:
1031:
1027:
1023:
1019:
1014:
1010:
1006:
1001:
996:
992:
988:
984:
980:
976:
971:
967:
963:
959:
955:
951:
947:
942:
938:
934:
930:
926:
922:
918:
912:
908:
904:
899:
894:
890:
886:
882:
878:
873:
869:
865:
860:
855:
851:
847:
843:
839:
835:
830:
826:
822:
817:
812:
808:
804:
800:
795:
791:
787:
782:
777:
773:
769:
765:
761:
757:
752:
748:
744:
740:
736:
732:
728:
723:
720:
719:
711:
707:
703:
699:
695:
691:
684:
681:
676:
672:
667:
662:
658:
654:
651:(2): 605–16.
650:
646:
645:Mol Cell Biol
642:
635:
632:
627:
623:
619:
615:
611:
607:
600:
597:
592:
588:
583:
578:
574:
570:
566:
559:
556:
551:
547:
543:
539:
535:
531:
524:
521:
516:
512:
508:
504:
500:
496:
489:
486:
481:
477:
472:
467:
463:
459:
455:
451:
447:
440:
438:
434:
429:
423:
415:
411:
407:
403:
399:
395:
391:
387:
380:
377:
370:
366:
363:
361:
358:
356:
353:
351:
348:
346:
343:
341:
338:
336:
333:
332:
328:
326:
320:
318:
312:
307:
305:
302:
297:
293:
291:
289:
285:
278:
276:
271:
269:
265:
261:
257:
251:
248:
245:
236:
232:
228:
221:
219:
212:
210:
208:
204:
194:
190:
188:
184:
180:
176:
175:
174:
172:
170:
160:
156:
152:
149:
145:
144:
140:
135:
132:
128:
127:
122:
121:
120:
118:
114:
110:
106:
102:
94:
89:
85:
81:
77:
73:
66:
64:
62:
58:
54:
50:
46:
42:
36:
34:
33:gene cassette
30:
26:
22:
18:
1025:
1021:
985:(21): e115.
982:
978:
949:
945:
920:
916:
880:
877:J. Mol. Biol
876:
841:
837:
806:
802:
763:
759:
733:(2): 87–92.
730:
726:
693:
689:
683:
648:
644:
634:
612:(2): 87–92.
609:
605:
599:
572:
568:
558:
536:(1): 52–69.
533:
530:J. Mol. Biol
529:
523:
498:
494:
488:
453:
449:
422:cite journal
397:
394:Biochemistry
393:
389:
385:
379:
324:
316:
308:Applications
300:
298:
287:
283:
272:
267:
255:
252:side-by side
249:
243:
241:
234:
230:
216:
200:
186:
168:
164:
158:
154:
147:
137:
130:
124:
108:
98:
88:transfection
83:
75:
52:
45:gene therapy
37:
20:
16:
15:
952:(1): 1–27.
898:10033/76653
809:(1): 7–28.
294:in parallel
266:, or other
155:integration
80:recombinase
1075:Categories
495:Biol. Chem
371:References
207:stem cells
143:resolution
57:expression
49:epigenetic
264:enhancers
213:Dual RMCE
126:inversion
113:crossover
41:transgene
1052:20953177
1009:12409474
966:23201421
937:21241707
907:19447116
868:19003073
825:14723844
790:16382151
747:14994271
710:22510960
675:16382151
626:14994271
591:21891781
550:20650281
515:11076013
480:16547094
450:Genetics
329:See also
178:isolated
139:deletion
105:monomers
1043:3576631
859:3476001
781:1346909
727:Genesis
666:1346909
606:Genesis
569:FASEB J
471:1526508
414:7947678
103:. Four
1050:
1040:
1007:
1000:135837
997:
964:
935:
905:
866:
856:
823:
788:
778:
745:
708:
673:
663:
624:
589:
548:
513:
478:
468:
412:
286:R and
279:after
185:this
171:sites
133:sites
93:clone
1048:PMID
1005:PMID
962:PMID
946:Gene
933:PMID
903:PMID
864:PMID
821:PMID
786:PMID
743:PMID
706:PMID
671:PMID
622:PMID
587:PMID
546:PMID
511:PMID
476:PMID
428:link
410:PMID
390:loci
256:loci
235:FRT-
136:the
123:the
53:loci
17:RMCE
1038:PMC
1030:doi
995:PMC
987:doi
954:doi
950:515
925:doi
921:407
893:hdl
885:doi
881:390
854:PMC
846:doi
811:doi
776:PMC
768:doi
735:doi
698:doi
694:109
661:PMC
653:doi
614:doi
577:doi
538:doi
534:402
503:doi
499:381
466:PMC
458:doi
454:173
402:doi
392:".
386:FRT
301:FRT
288:att
284:att
268:cis
244:FRT
169:FRT
159:FRT
148:FRT
131:FRT
109:FRT
84:FRT
1077::
1046:.
1036:.
1024:.
1020:.
1003:.
993:.
983:30
981:.
977:.
960:.
948:.
931:.
919:.
901:.
891:.
879:.
862:.
852:.
842:50
840:.
836:.
819:.
805:.
801:.
784:.
774:.
764:26
762:.
758:.
741:.
731:38
729:.
704:.
692:.
669:.
659:.
649:26
647:.
643:.
620:.
610:38
608:.
585:.
573:25
571:.
567:.
544:.
532:.
509:.
497:.
474:.
464:.
452:.
448:.
436:^
424:}}
420:{{
408:.
398:33
396:.
262:,
1054:.
1032::
1026:7
1011:.
989::
968:.
956::
939:.
927::
909:.
895::
887::
870:.
848::
827:.
813::
807:6
792:.
770::
749:.
737::
712:.
700::
677:.
655::
628:.
616::
593:.
579::
552:.
540::
517:.
505::
482:.
460::
430:)
416:.
404::
290:L
150:s
141:/
19:(
Text is available under the Creative Commons Attribution-ShareAlike License. Additional terms may apply.
