Recombination based transformation technologies

Recombination based transformation technologies

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 geneTrait 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:
1.Crossing lox plant with cre-expressing plant to obtain F1, which will be expected to undergo Cre-lox
recombination
2.Retransform the lox plant with cre gene.
3.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
Trait gene
loxP loxP
Chemical
-induced
cre gene
chemical
Trait gene
loxP

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

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

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

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

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

8. Resolving complex integration pattern

9. Enhancing plastid transformation rate with phiC31 system
Lutz et al. (2004) Plant J. 37(6):906-13
phiC31 system: Recombination sites: attP, attB, attL, attR
Recombinase: phiC31recombinase
attPXattB
phiC31 recombinase
attRXattL
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.

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