1. 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

2. 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

3. 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

4. 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

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

6. 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

7. 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

8. Resolving complex integration pattern

9. 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

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