Recombination based transformation technologies Removal of selection marker gene Gene integration into dedicated genomic sites Conversion of multi-copy locus to single-copy locus Enhancing plastid transformation rates Zinc Finger Nuclease mediated gene integration Transposing transgene from complex locus to new genomic sites to generate single-copy insertions

Marker removal Marker gene Trait gene loxP loxP CRE Trait gene + loxP Since a loxP- flanked DNA fragment is deleted upon introduction of Cre activity into the nucleus, marker removal can be accomplished by designing transformation construct that contain loxP flanked marker gene. There are following ways to introduce Cre activity into the transgenic plants containing the marker gene: Crossing lox plant with cre-expressing plant to obtain F1, which will be expected to undergo Cre-lox recombination Retransform the lox plant with cre gene. Use inducible cre gene embedded into the lox construct. The Cre activity can be induced by applying inducer to initiate the recombination which will lead to self-excision of cre and the marker gene (see below) Marker gene Chemical-induced cre gene Trait gene Trait gene chemical loxP loxP loxP

Genomic targeting with a positive-selection lox integration vector Cre-lox mediated gene integration (targeting) was first demonstrated in mammalian cells Genomic targeting with a positive-selection lox integration vector allows highly reproducible gene expression in mammalian cells lacZ (with or without enhancer) Cmv pro neo Cre Cmv pro= cytomegalo virus promoter lacZ Cmv pro neo Fukushige and Sauer (1992) PNAS 89: 7905

Plasmid Lines parent1 parent2 -galactosidase activity Plasmid Lines parent1 parent2 1 2 3 4 5 6 7 21.5 20.5 18.5 27 12.5 56 23 28 32 34 25 0# #1 without enhancer 1 2 3 4 5 6 7 8 285 210 195 235 157 331 267 367 141 120 162 390* 139 111 217 174 #2 with enhancer *illegitimate integration of second intact copy #deletion of lacZ gene Fukushige and Sauer (1992) PNAS 89: 7905

Recombinase mediated cassette exchange Requirement: a pair of hetero-specific recombination (lox) sites Feng et al. J. Mol. Biol., 1999, 292: 779

FLP-mediated DNA mobilization to specific target sites in Drosophila chromosomes: excision and re-integration strategy C A C Pro Pro A FLP B D FLP B Target Donor D Empty donor Integrant Upto 5% efficiency of germline integration in Drosophila Golic et al., 1997, Nucl. Acid Res. 25: 3665

Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Displacement strategy Transient expression strategy hpt hpt p35S-cre 35S Cre 35S luc hpt Cre 35S luc 35S hpt Albert et al. 1995, Plant J. 7: 649

Resolving complex integration pattern

Enhancing plastid transformation rate with phiC31 system phiC31 system: Recombination sites: attP, attB, attL, attR Recombinase: phiC31recombinase phiC31 recombinase attP X attB attR X attL PhiC31 + Xis factor Therefore, phiC31 system can be used as a dedicated integration system (reversion would not occur in the absence of Xis. Lutz et al propose that this system could be integrated into plastid genome of plant species for which plastid transformation rates are very low. They assume that low transformation rate is based on low homologous recombination rates in the plastids of these plant species (all except tobacco). If integration was dependent on phiC31 system, then plastid transformation rate could possibly go up. However Lutz et al simply tested the feasibility of phiC31 system in tobacco and not in any other plant species. Lutz et al. (2004) Plant J. 37(6):906-13

Transposon mediated single copy gene delivery leads to increase transgene expression stability. Koprek et al 2001, Plant Physiol. 125:1354