Recombination based transformation technologies
Marker removal
Gene targeting
Resolving complex locus
Enhancing plastid transformation rates
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:
1.
2.
3.
Crossing lox plant with cre-expressing plant to obtain F1, which will be expected to undergo Crelox 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
loxP
Chemical
-induced
cre gene
Trait gene
loxP
Trait gene
chemical
loxP
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
Fukushige and Sauer (1992) PNAS 89: 7905
neo
-galactosidase activity
Plasmid Lines
1
2
3
4
5
6
7
#1
without
enhancer
#2
with
enhancer
1
2
3
4
5
6
7
8
parent1
parent2
21.5
20.5
18.5
27
12.5
56
23
28
32
28
34
25
25
0#
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
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
C
Pro
A
Pro
D
Target
FLP
Donor
FLP
B
A
D
Integrant
B
Empty donor
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.
Transient expression strategy
Displacement strategy
p35S-cre
35S
35S
Cre
hpt
35S
Cre
Albert et al. 1995, Plant J. 7: 649
35S
luc
hpt
luc
Site-specific integration of T-DNA in Arabidopsis
mediated by Cre recombinase
Both transient expression strategy and displacement
strategy were used.
Both gave low site-specific integration frequency.
Resolving complex integration pattern
Srivastava et al. (1999) PNAS 96: 11117
Enhancing plastid transformation rate with phiC31 system
phiC31 system: Recombination sites: attP, attB, attL, attR
Recombinase: phiC31recombinase
attP
X
attB
phiC31 recombinase
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