Acta Botanica Sinica
植 物 学 报
2004, 46 (7): 862－866
http://www.chineseplantscience.com
A New Cre/lox System for Deletion of Selectable Marker Gene
YUAN Yuan, LIU Yun-Jun, WANG Tao*
(State Key Laboratory for Agrobiotechnology, China Agricultural University, Beijing 100094)
Abstract: A new inducible Cre/lox system was constructed in transgenic tobacco (Nicotiana tabacum
L.) plants. The inducer-treatment of tobacco callus mediates an excision event in which the selectable
marker gene and Cre gene between two lox sites were deleted. A chloroacetanilide-induced promoter
(In5-2) was used to control the expression of Cre gene in this system. Molecular analysis of transgenic
tobacco plants showed the interested gene, β-glucuronidase (gus), was integrated into the genome whether
removing has been successful, and forty-five out of forty-eight T0 plants were transgenic tobacco without
the marker gene, hpt. This system uses a single vector to circumvent the flaw of other dual recombinase
vector systems.
Key words: chloroacetanilide-induced promoter (In5-2); Cre/lox; marker-free; transgenic tobacco
Since the first transgenic plant was created in 1983, a lot
of transgenic crops, such as tobacco, tomato, maize, cotton,
etc., have come into the world carrying the external genes
(David, 2000). Genetic transformation is becoming an important tool in obtaining transgenic plants. The marker gene,
a necessary portion of the plant transformation vector, is
used for selection of transformants. Antibiotic or herbicide
resistance genes account for the majority of selectable
markers used (Yoder et al., 1994). Marker genes were also
integrated into plant genome together with the target gene,
and some problems produced by marker genes are greatly
noticed (Elena et al., 2000). For example, producing herbicide-resistant weeds by cross-pollination; threatening biology diversity and the environment; the theoretical risk in
food products; appearance of transgenic silence due to
multiple copies of the same selectable marker gene, etc.
In the field of plant genetic engineering, transgenic
biosafety is one of the significant problems. From the end
of the 1980s, the deletion of selectable marker genes by the
Cre/lox system from the P1 phage was reported. There are
three developmental approaches: (1) simple deletion: Cre
gene was introduced into plants and only the marker gene
was deleted by sexual crossing or secondary transformation with long generation times (Odell et al., 1990; Dale
et al., 1991; Bayley et al., 1992; Russell et al., 1992; Andrew
et al., 1999). While, although the marker gene was deleted,
the introduced Cre gene would bring some biosafety
problems. In addition, this technology is a time-consuming
and hard work with lower efficiency; (2) deletion and other
manipulation: use the Cre/lox system as a tool to study
single-copy transgenic plants, gene clone technology and
site-specific integration, etc., going with the deletion of
marker genes. (Cydne et al., 1993; Odell et al., 1994; Vergunst
et al., 1998; Que et al., 1998; Liu et al., 1998; Sivastava
et al., 2001); (3) induced deletion: marker gene and Cre
gene were deleted by a chemical-induced system with only
once transformation (Zuo et al., 2001). In this phase no
new gene was introduced into the plant and the method
has saved time and has high efficiency.
The new Cre/lox system reported here consists of a
maize Chloroacetanilide-induced promoter (In5-2), Cre
gene, two lox sites with the same orientation, β-glucuronidase (gus) gene used as the interested gene and hpt
gene used as the selectable marker gene. In this study, Cre
gene and hpt gene were linked between the two same oriented lox site. When the inducer was added into the callus,
the recombined reaction occurred, so hpt gene and Cre
gene were deleted simultaneously. These results suggested
that the new Cre/lox system would be effective for producing marker-free transgenic plants.
1 Materials and Methods
1.1 Materials
Nicotiana tabacum L., bacteria of Agrobacterium
tumefaciens strain LBA4404, as well as plant transformation vector p1301 were used in this study.
1.2 Construction of plant transformation vector
DNA manipulations were performed essentially as described by Sambrook et al. (1989). The chimeric Cre gene
came from pETCre (kindly provided by Dr. LIN Zhong-Ping,
Received 3 Feb. 2004 Accepted 29 Apr. 2004
Supported by the National Special Program for Research and Industrialization of Transgenic Plants (JY03-B-18-01) and the State Key
Basic Research and Development Plan of China (2001CB1090).
* Author for correspondence. E-mail: <[email protected]>.
YUAN Yuan et al.: A New Cre/lox System for Deletion of Selectable Marker Gene
College of Life Sciences, Peking University) under control
of the maize In5-2 promoter (Yuan et al., 2004). The lox
sites were supplied as synthesized oligo adapters by
Sangon company, Shanghai. The hpt gene driven by
CaMV35S promoter and Cre gene were inserted between
the two lox sites. The whole DNA fragment containing lox,
hpt and Cre was ligased into sites of p1301 digested with
XhoⅠ/HindⅢ, to produce plasmid pNC5 (Fig.1).
1.3 Plant transformation and chemical induction
B i na r y v ec t o r p N C 5 wa s t r a n s f o r me d in t o
Agrobacterium tumefaciens LBA4404. Transgenic tobacco
plants were produced using the leaf-disc transformation
procedure (Horsch et al., 1985). The transformed tissues
were selected by Hygromycin B (200 mg/L), and cefotaxime
(500 mg/L) was used to remove the bacteria.
The inducer 2-chloro-N-(methylaminocarbonyl)
benzenesulfonamide was used to induce the expression of
Cre gene. The transformed tobacco leaf-discs were selected
on the solid MS medium containing 200 mg/L hygromycin
for 30 d. Then explants were transplanted to the solid MS
medium containing 0.2 g/L inducer and induced for 15 d.
1.4 PCR analysis of transgenic plants
Isolation of genomic DNA from leaf material was performed essentially as described previously (Harrison et al.,
1997). PCR was performed in GeneAmp 9700 with primers
respectively forGUS: (G1,5'-GGTGGGAAAGCGCGTTACAAG-3'; G2, 5'-GTTTACGCGTTGCTTCCGCCA-3'); for In5-2
promoter: (I1, 5'-GGCGTTGAGCCTTTTTCTAC-3'; I2, 5'TGTTTCCTGCTACTCGTTGG-3'); for hpt: (H1, 5'G G C GAAG AAT C T C G T G C T T T C A- 3 '; H 2 , 5 'CAGGACATTGTTGGAGCCGAAA-3'); for Excision: (N1,
5'-CTTAATAACACATTGCGGACGT-3'; N2, 5'-AGAGGCGGTTTGCGTATT-3').
1.5 Southern blotting analysis of transgenic plants
Genomic DNA was extracted from 2 g of leaf tissue using the SDS procedure (Harrison et al., 1997). Five µg genomic DNA was digested with restriction endonuclease
(NcoⅠ /NheⅠ, or SalⅠ, or EcoRⅠ), electrophoresed in a
0.8% agarose gel and transferred onto Hybond N+ nylon
membrane following the manufacturer’s recommendations
for alkaline transfer. Probes were prepared from DNA fragments eluted from agarose gels using DNA Recovery Kit
(BioDev), and labeled using Random Primer DNA Labeling
Kit (Promaga) incorporating [α-32P] dCTP. Standard procedures
were used for DNA blot analysis (Sambrook et al., 1989).
2
Results
2.1 Constructions of vectors
The vector pNC5 was constructed to transform tobacco
８６３
plants. In pNC5, Cre gene and hpt gene were located between the two lox sites with the same orientation. Cre gene
was controlled by maize In5-2 promoter and would not express without the inducer. At the beginning, hpt was used
to select the transgenic shoots. The inducer was added to
the medium after selection, and the Cre gene was induced
to express Cre recombinase that had the effect on the two
lox sites with the same orientation. So the recombination
occurred and hpt gene and Cre gene between lox loci was
deleted (Fig.1). Control of Cre gene expression is an essential step in stabilization of recombination events, because
presence of Cre recombinase before site-specific deletion
will lead to subsequent loss of the selectable marker gene
that would lead to enable selection of transgenic plants. In
this study, the deletion reaction itself would lead to absence of Cre gene that was between lox loci. So the Cre
gene was not introduced into the plant genome.
2.2 Transformation of tobacco with plasmid pNC5
The leaf discs of N. tabacum were transformed with A.
tumefaciens LBA4404 containing pNC5 and cultured on
hormone-free MS medium in the dark. After 3 d, the leaf
discs were moved to the sprout culture medium with 200
mg/L Hygromycin. Some buds were differentiated within
one month. Naught point two g/L inducer was added in
solid MS sprout culture medium for 15 d (Yuan et al., 2004).
2.3 Verification of marker-free transgenic tobacco by
molecular analysis
The results of PCR analysis with GUS primers showed
that we obtained total forty-eight induced transgenic tobacco plants and ten un-induced transgenic tobacco plants.
These transgenic tobacco plants were analyzed by the PCR
method to confirm the presence or absence of the hpt gene,
gus gene and In5-2 promoter. Wild-type tobacco was used
as control. In Fig.1, the position of the primer binding sites
and the expected sizes of amplified fragments are depicted.
The predicted 0.5 kb hpt fragment, 1.2 kb gus gene fragment and 1.5 kb In5-2 promoter fragment were amplified in
un-induced transgenic tobacco with primers H1-H2, G1-G2
and I1-I2, respectively. In the induced transgenic tobacco
plants, the hpt and In5-2 fragments disappeared, but the
predicted 1.2 kb gus gene fragment and 0.4 kb fragment
were amplified with primers G1-G2 and N1-N2, respectively
(Fig. 2). The results showed that excision event occurred in
the transgenic tobacco plants.
We performed genomic Southern blotting analysis to
test whether the DNA fragment was excised in the tobacco
genome. A gus probe detected an NcoⅠ/NheⅠ fragment
with 2 kb expected size in all induced or un-induced
transgenic tobacco plants (Fig.3A). Using hpt (Fig.3B) and
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Fig.1. A schematic diagram of the pNC5 vector and chemical-induced DNA excision. A. Schematic map of the transformation vector
pNC5. bolded box, the hygromycin resistance (hpt) gene. Box, β-glucuronidase (gus) gene; arrow with italic line, maize In5-2 promoter;
bolded arrows, lox site; box with italic line, Cre recombinase gene. H1, H2, I1, I2, G1, G2, N1 and N2, primers. XhoⅠ, ApaⅠ, EcoRⅠ,
BglⅡ and SalⅠ, recognition sequences for restriction enzymes. B. Excisable progress of marker gene and Cre gene. C. Schematic map
of the vector p1301. LB, RB, left and right border fragments of Agrobacterium.
In5-2 (Fig.3C) coding sequence as probes respectively, no
hybridization signal was detected in recombinant plants,
whereas a 2.3 kb 35S-hpt-nos fragment and a 2.5-kb DNA
fragment including In5-2 promoter and Cre gene of the
expected size was present in un-induced transgenic plants.
This was diagnostic for precise site-specific excision after
chemical inducing.
2.4 Recombination efficiency after chemical inducing
The intention of recombination is to delete the selectable marker gene from the genome of transformants with
gus gene in this paper. Forty-eight induced transgenic tobacco plants contained gus gene and among these only
three plants contained hpt gene, which indicated that the
efficiency of inducing Cre-catalyzed excision was 93.8%.
3 Discussion
We have constructed a new Cre/lox system with high
effectiveness, in which Cre gene was under the control of
a Chloroacetanilide-induced In5-2 promoter, to generate
marker-free transgenic plants. Cre recombinase mediated
the elimination of selectable marker gene and Cre gene.
Some papers have also discussed the efficiency of Cremediate excision effect. The excision ratio was 53.8% (42/
78) in pollination (Dale et al., 1991), 94.7% (36/38) in secondary transformation (Russell et al., 1992), 100% (19/19)
in CLX system (Zuo et al., 2001). In contrast with these
Cre/lox systems, our system had higher efficiency in the
excision of the heterogenous gene and the excision ratio
was 93.8%. The fact was resultant because of the chemicalinduced promoter. Taking one with another, transcripts
activity of the induced promoter was lower than constitutive promoter such as CaMV35S promoter. So, expression
of Cre gene could be a little weakened and the efficiency of
recombination declined. The best method to heighten efficiency is to use the stronger promoter. But the number of
isolated induced-promoters with high transcript activity
was small. One of the important tasks is to clone stronger
induced promoters, in order to regulate expression of gene
either in laboratory or in field in the future (Reynolds, 1999).
Unlike the Cre/lox system described previously (Chen
et al., 2003), our system has several characteristics: first,
after induced DNA deletion, all unnecessary components
including Cre gene and selectable marker gene would be
moved from transgenic plants’genome; second, progeny
separation would not bring the genetic problem and the
deletion event was needed only one time; third, this
YUAN Yuan et al.: A New Cre/lox System for Deletion of Selectable Marker Gene
Fig.2. PCR analysis of transgenic tobacco plants. A. gus. B. hpt.
C. In5-2 promoter. D. Excision. M, 1 kb DNA Ladder; 1, untransgenic tobacco plants as negative control; 2, plasmid pNC5
(A, B, C), plasmid p3301 (D); 3－6, induced transgenic tobacco
plants; 7－10, un-induced transgenic tobacco plants.
system would not affect the conventional transformed
method; fourth, the structure of plant transformation vector and manipulation is simplified; fifth, expression of Cre
gene could be induced at any given time, which is of the
utmost importance for the eliminating biosafety problem of
transgenic plants that the selectable marker gene generated,
and for the generation of transgenic plants carrying multiple transgenes.
In further studies, this system can also be used for deletion of the interest gene in the field or in the laboratory
because the inducer (Chloroacetanilide) is a kind of herbicide safer and used in agriculture (Repasi et al., 1995). The
biosafety problem of heterogenous gene, such as Bt gene,
has also been made known to people. How to eliminate
negative effects of these gene will become one new significant work. And there are some non-transgenic plants from
transgenic plants (Keenan, 2002) by induced Cre/lox system in future.
Acknowledgements: We thank Dr. WANG Min (College
８６５
Fig.3. Southern blotting analysis of transgenic tobacco plants.
A. gus probe. B. hpt probe. C. In5-2 probe. Genomic DNA was
hybridized with gus probe (A), In5-2 probe (B) and hpt probe
(C), re spectivel y. 1，pNC5 as positive cont rol; 2,
untransformed tobacco plant; 3－9, uninduced transgenic plants;
10－16, induced transgenic tobacco plants.
of Sciences, China Agricultural University) for providing
the inducer. And we are grateful to Dr. LIN Zhong-Ping
（College of Life Sciences, Peking University）for his generous gifts of the Cre gene.
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