Site-specific T-DNA integration in Arabidopsis thaliana mediated by the...

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This article has been accepted for publication and undergone full peer review but has not been through the copyediting, typesetting, pagination and proofreading process which may lead to differences between this version and the Version of Record. Please cite this article as an 'Accepted Article', doi: 10.1111/tpj.12202 This article is protected by copyright. All rights reserved.

Received Date : 06-Jun-2012

Revised Date : 04-Apr-2013

Accepted Date : 08-Apr-2013

Article type : Technical Advance

Site-specific T-DNA integration in Arabidopsis thaliana mediated by the combined

action of CRE recombinase and φC31 integrase

Annelies De Paepe, Sylvie De Buck, Jonah Nolf, Els Van Lerberge and Ann Depicker*

Department of Plant Systems Biology, VIB, Technologiepark 927, B-9052 Gent, Belgium and

Department of Plant Biotechnology and Bioinformatics, Ghent University, Technologiepark

927, B-9052 Gent, Belgium

* Correspondence (Tel +32(0)3313940; fax +32(0)3313940; email [email protected]

ugent.be)

E-mail adresses authors:

Annelies De Paepe: [email protected]

Sylvie De Buck: [email protected]

Jonah Nolf: [email protected]

Els Van Lerberge: [email protected]

Ann Depicker: [email protected]

Running title: Site-specific T-DNA integration in Arabidopsis

Keywords: T-DNA, site-specific integration, φC31 integrase, CRE recombinase, gene

stacking, Arabidopsis.

SUMMARY

Random T-DNA integration into the plant host genome can be problematic for a variety of

reasons, including potentially variable transgene expression as a result of different

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This article is protected by copyright. All rights reserved.

integration positions and multiple T-DNA copies, the risk of mutating the host genome and

the difficulty of stacking well defined traits. Therefore, recombination systems have been

proposed to integrate the T-DNA at a pre-selected site in the host genome. Here, we

demonstrate the capacity of the φC31 integrase (INT) for efficient targeted T-DNA integration.

Moreover, we show that the iterative site-specific integration system (ISSI), which combines

the activities of the CRE recombinase and φC31 integrase, allows to target genes to a pre-

selected site with concomitant removal of the resident selectable marker. To begin, plants

expressing both the CRE and INT recombinase and containing the target attP-site were

constructed. These plants were supertransformed with a T-DNA vector harboring the loxP

site, the attB-sites, a selectable marker and an expression cassette encoding a reporter

protein. Three out of 35 obtained transformants (=9%) showed transgenerational site-

specific integration (SSI) of this T-DNA and removal of the resident selectable marker, as

demonstrated by PCR, Southern blot, and segregation analysis.

In conclusion, our results show the applicability of the ISSI system for precise and

targeted Agrobacterium-mediated integration, allowing the serial integration of transgenic

DNA sequences in plants.

INTRODUCTION

The widely used Agrobacterium plant transformation method allows efficient genetic

modification of many plants species, but still has some drawbacks. Due to a lack of control

on the inserted copy number and the site of integration, the transgenics often harbor multiple

copies of the transgene at one or more random genomic locations (Jones et al., 1987;

Hobbs et al., 1993; Cheng et al., 1997; De Neve et al., 1997; De Buck et al., 1999; De Buck

et al., 2009). Variations in copy number and complex transgene integration events have

been correlated with transgene expression variability and instability caused by post-

transcriptional and transcriptional transgene silencing (Matzke et al., 2000; Muskens et al.,

2000; Wang and Waterhouse, 2000; De Buck et al., 2001). Also, random integration of the

transgenes into the host plant genome can be problematic for a variety of reasons, including

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potential variable transgene expression resulting from position effects and the risk of

mutating the host genome during transgene integration (Matzke and Matzke, 1998). As a

consequence, often hundreds of transformants must be screened to find an elite event

conferring good stable transgene expression and uncompromised agronomic performance.

Therefore, more precise technologies are highly desirable for the production of

transgenic crop plants with predictable transgene integration sites, and also for addressing

issues in regulatory approval and social acceptance. Furthermore, the challenge for future

crop improvement depends on the ability to stack multiple transgenic traits at a

predetermined integration site in a single-copy fashion. Recycling of marker genes in

successive transformations is therefore also essential as not enough efficient and approved

selectable markers are available.

Site-specific recombination methods have been developed to remove selectable

markers to produce marker-free transgenic plants and to integrate the transgene into an

appropriate genomic target (Wang et al., 2011 and references therein; Tuteja et al., 2012),

as well as to reduce the number of transgene copies (Srivastava and Ow, 2001; De Buck et

al., 2007; De Paepe et al., 2009). The Cre/loxP, R/Rs and FLP/FRT recombinases from

bacteriophage P1 of Escherichia coli (Dale and Ow, 1990; Odell et al., 1990; Russell et al.,

1992), from the SR1 plasmid of Zygosaccharomyces rouxii (Onouchi et al., 1991; Onouchi et

al., 1995) and from the plasmid of Saccharomyces cerevisiae (Lyznik et al., 1993; Kilby et

al., 1995), respectively, have been the focus of multiple studies in plants and other

organisms. These site-specific recombination systems are working efficiently and are simple

to use; however they work bidirectionally, meaning that these recombinases catalyze freely

reversible reactions and are therefore less ideal for gene stacking. Unwanted events such as

deletion of previously introduced DNA cannot be prevented because fully functional

recombination sites are needed for subsequent gene targeting of other DNA fragments. In

contrast, the integrase from the Streptomyces phage φC31 (Thorpe and Smith, 1998), a

member of the serine family of recombinases, catalyzes the recombination reaction of the

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non-identical attB and attP sites of φC31 in only one direction to generate attL and attR sites.

This feature gives the φC31 system a distinct advantage as a tool for site-specific DNA

integration (Smith et al., 2010). In plants, the φC31 integrase was applied for stable plastid

transformation (Lutz et al., 2004), marker gene excision in tobacco (Ow, 2002;

Kittiwongwattana et al., 2007), removal of transgene fragments in Arabidopsis (Gils et al.,

2008; Thomson et al., 2010), chromosomal recombination in Arabidopsis (Ow, 2002),

intrachromosomal inversion and transgene excision in wheat (Rubtsova et al., 2008; Kempe

et al., 2010), and φC31 integrase-mediated excision in barley (Kapusi et al., 2012). More

recently, the use of the Bxb1 integrase, a recombinase from phage Bxb1 analogous to φC31,

was described for site-specific integration with 10% efficiency in tobacco (Ow, 2011; Yau et

al., 2011) and for site-specific excision in Arabidopsis (Thomson et al., 2012).

The combined use of multiple site-specific recombination systems to direct DNA

integration and concomitant marker gene removal to produce marker-free targeted site-

specific integration events has been proposed and demonstrated in previous studies

(Srivastava and Ow, 2001; Ow, 2002; Akbudak and Srivastava, 2011; Wang et al., 2011;

Nandy and Srivastava, 2012). Furthermore, the combination of a non-reversible with a

reversible recombination system permits efficient sequential gene stacking of many

transgenes.

Brown and co-workers established a method in chicken DT40 cells that enables the

iterative integration of transgenic DNA sequences by combining the site-specific

recombinase activities of the unidirectional φC31 integrase and the bidirectional CRE

recombinase (Dafhnis-Calas et al., 2005). This method, termed iterative site-specific

integration (ISSI), allows to stack genes at a pre-selected site with concomitant removal of

the resident selectable marker. Also another member of the serine recombinase family, the

φBT1 integrase, in combination with the CRE recombinase, was shown to promote targeted

iterative integration of transgenic DNA in vertebrate chromosomes (Xu et al., 2008). Using

this method, four successive cycles of gene stacking were demonstrated in vertebrate cells

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and five serial integration cycles were described in mammalian cells (Dafhnis-Calas et al.,

2005; Xu et al., 2008).

We investigated the potential of the ISSI strategy as a tool for iterative integration of

transgenic DNA after Agrobacterium-mediated floral dip transformation in Arabidopsis.

Therefore, we developed two T-DNA vectors, i.e. the T-DNA target vector and the T-DNA

targeting vector. The former contains an attP target site for subsequent integration of

targeting T-DNAs and a loxP site for removal of the selectable marker by the CRE

recombinase upon integration of the targeting T-DNA.; the latter contains two directly

oriented loxP sites inside the T-DNA borders needed for circularization; subsequent site-

specific integration is mediated by φC31 between one of its two attB sequences and the

resident attP sequence.

Here, we describe the first cycle of the ISSI strategy in Arabidopsis consisting of site-

specific T-DNA integration (SSI) at the attP target site and excision of the resident marker

construct in a single step. We also demonstrate that site-specific T-DNA integration occurred

in the progeny of 9% of the obtained T-DNA transformants.

RESULTS

Experimental design to demonstrate the feasibility of the ISSI strategy

In the presented ISSI strategy, an Arabidopsis thaliana ecotype C24 transformant was

selected, constitutively expressing the CRE and INT recombinases, and is referred to as the

parental transgenic line, Cre-Hsc1 (Figure 1a). This target line contains two T-DNAs, i.e. the

Hsc1 T-DNA and the Cre T-DNA. The Hsc1 T-DNA contains a loxP site (Figure 1a, open

triangle) at the left and an attP (PP’ box) site at the right side of a selectable hygromycin

(Hyg) resistance marker gene. The loxP site allows removal of the selectable marker by the

CRE recombinase upon integration of the targeting T-DNA, while the PP’ site serves as the

target for site-directed integration. Besides this, the Hsc1 T-DNA contains the Pcas driven

φC31 integrase (INT). The Cre-Hsc1 line also harbors the unlinked Cre T-DNA with the

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biolaphos resistance marker (BAR) and the 35S promoter driven CRE gene expression

cassette.

To demonstrate the targeted stacking of a gene of interest (GOI), a dedicated

targeting vector, pK2L2B (Figure 1b), was constructed. This targeting T-DNA vector contains

two directly oriented loxP sites just between the T-DNA borders to obtain a double-stranded

circular intermediate from the linear T-DNA by CRE-mediated recombination (Figure 1c1). In

between those loxP sites, this targeting vector contains the neomycin phosphotransferase II

(NPTII) selectable marker and a 35S promoter driven enhanced green fluorescent protein/β-

glucuronidase (eGFP/GUS) fusion gene as GOI to stack in the PP’ site in the target line. The

eGFP/GUS gene is flanked by two copies of an attB sequence, BB’I and BB’II (Figure 1b).

The INT-mediated recombination may occur between the attP site and either one of the two

attB sites present in the targeting vector, leading to two different intermediate recombination

products. The INT-produced recombined intermediate I (Figure 1c2) is obtained by INT

recombination between the PP’ and BB’I site (Figure 1c1). CRE-mediated recombination

between the in tandem oriented loxP sites surrounding the resident HPT selectable marker

finally results in a targeted insertion of the T-DNA and concomitant removal of the selectable

marker (Figure 1d). Thus, this SSI transformant can be used for a next round of INT-

mediated site-specific recombination, as it contains a single attB target site. The other

possible INT-produced recombined intermediate II (Figure 1c3), obtained by INT

recombination between PP’ and BB’II, however contains the eGFP/GUS gene together with

the HPT selectable marker between the two loxP sequences, and thus CRE recombination

deletes the transiently inserted GOI, resulting in recombined outcome II as shown in Figure

1e. Half of the INT-mediated recombination products is thus not useful for ISSI and can be

distinguished from recombined outcome I by PCR or Southern blot analysis (see further).

We have illustrated the ISSI strategy in Figure 1 as though the site-specific

integration reaction occurred first by INT after CRE-mediated circularization of the targeting

vector, but ISSI can also be obtained by integration of the targeting T-DNA circle by CRE

into the genomic loxP site of the Hsc1 T-DNA followed by the INT recombination for the

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excision reaction (Figure S1). However, the order in which the recombinases act does not

make a difference in the final and stabilized outcome.

Construction of the φC31 and CRE recombinase-expressing target line Cre-

Hsc1 with attP target site

Homozygous CRE-expressing Arabidopsis plants (Marjanac et al., 2008) were transformed

with the Hsc1 T-DNA vector (De Paepe et al., 2009) (Figure 1a) by floral dip to obtain a

single-copy Cre-Hsc1 target line suitable for ISSI recombination reactions. The obtained T1

Cre-Hsc1 transformants grew normally and did not show any clear phenotypic alterations,

and single-copy lines were selected by Southern blot analysis (De Paepe et al., 2009). Leaf

extracts of the T1 transformants containing a single Hsc1 T-DNA copy were tested for the

expression of the φC31 integrase by immunoblot analysis with an anti-φC31 integrase

antibody. We selected one single-copy target Cre-Hsc1 transformant with a good φC31

integrase expression. This T1 plant was selfed and seven T2 Hyg-resistant offspring plants

(Table 1- Recipient plants 4, 7, 13, 15, 16, 19 and 23), homozygous (Ho) or hemizygous (He)

for the Hsc1 T-DNA were used for floral dip transformation with the pK2L2B targeting vector.

Creation of SSI events and selection of transformants

The pK2L2B targeting construct as shown in Figure 1b was used for transformation into the

seven Cre-Hsc1 target plants to evaluate how many targeted integration events versus

random insertions would be recovered. In fact, only the former are expected, as in case of

random integration, resolvement of the internal T-DNA segment with the kanamycin (Km)

selectable marker between the loxP sites would leave behind only the LB-loxP-RB footprint

at the integration site. The seeds obtained after floral dip of the T2 Cre-Hsc1 plants with the

pK2L2B targeting construct are indicated as the R1 seed stocks. Selection of R1 seedlings

on Km yielded a total of 35 transformants from about 14,000 seeds, corresponding to an

average transformation frequency of 0.25% (Table 1). In parallel, transformation of the same

vector in CRE-expressing plants without a target attP recombination site generated five Km-

resistant plants out of 4,000 seeds (=0.13%) (Table 1). This shows that pK2L2B

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transformants can be obtained in which the T-DNA is randomly inserted and not excised by

the CRE recombinase, for instance due to deleted or inaccessible loxP sites. Thus, among

the 35 R1 transformants, a fraction of those is expected to result from random T-DNA

integration concomitant with maintaining the Km selectable marker. Also, C24 wild-type

plants were transformed with the same targeting vector for estimating the random integration

efficiency of the targeting vector. Here, the transformation efficiency was 0.45%.

Molecular analysis of the obtained R1 recombinants

Leaf DNA of the 35 R1 Km-resistant plants was PCR analyzed with primer sets a/b and a/c

(Figure 1d,e). Amplification of a fragment of 478 bp (primers a/b) or 1022 bp (primers a/c)

indicates a targeted recombined outcome I or II, respectively. The diagnostic fragment of

478 bp was found in three and that of 1022 bp in six of the 35 R1 transformants (Table 1).

These nine recombination products were derived from four recipient Cre-Hsc1 plants (4, 7,

13 and 16) while no positive PCR recombination products were seen in the R1 transformants

of the other three Cre-Hsc1 recipient plants, nor in the Km-resistant Cre-1 and C24

transformants. The nine obtained PCR fragments were sequenced and the results confirmed

precise site-specific recombination for recombined outcomes I and II in the leaf DNA of R1

transformants (Table 1). Subsequently, these nine R1 Km-resistant transformants with a

scored SSI event in the leaves were selfed for analysis of the R2 seed stocks.

Segregation analysis to investigate germ line transmission of the SSI event

To determine whether the recombined outcome products were heritably transmitted to the

progeny and whether the resident selectable HPT marker was deleted, we examined the

segregation of the R2 progenies of the nine R1 SSI PCR positive transformants on Km- and

Hyg-containing medium. Surprisingly, several of those seed stocks showed a very low

number of Km-resistant seedlings, as compared to the expected 75% frequency (Table 2).

Also unexpected was that R2 progeny plants of most transformants are still 100% Hyg

resistant. This result indicates that the SSI event demonstrated in the leaves of the R1

transformants was not transmitted through the germ cells and also that the targeting Km-

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resistant T-DNA was lost from many of the germline precursor cells in several of the R1

transformants. This is probably due to resolvement of the in tandem oriented loxP-sites in

the targeting T-DNA after random integration.

We further reasoned that the SSI event could take place in the germline progenitors

in kanamycin resistant R2 plants. Therefore, some of the Km-resistant R2 plants from each

transformant were selfed and the obtained R3 seed stocks were again analyzed for the

segregation of the targeting Km-resistant and the target Hyg-resistant constructs. As an SSI

T-DNA allele should be correlated with the loss of the Hyg resistance marker at the

integration locus, all R3 SSI progeny plants derived from a hemizygous target plant should

be 100% sensitive to Hyg and 100% or 75% resistant to Km. On the other hand, SSI R3

progeny plants derived from a homozygous Hsc1 locus plant should segregate (3:1) on Hyg

and Km, or should be 100% sensitive to Hyg and 100% resistant to Km.

We found that all R3 progeny plants of the 13-3, 16-11 and 16-12 R2 transformants had

lost Hyg resistance while being 100% Km resistant, or showed Hyg and Km resistance in the

predicted segregation ratio (3:1) (Table 2). In the other tested R3 seed stocks (4-1, 4-2, 7-1,

16-3 and 16-4) the segregation pattern on Hyg and Km was indicative for random integration

of the non-excised T-DNA targeting construct. Indeed, these R3 seed stocks derived from

different R2 transformants were fully Hyg resistant, or showed unlinked segregation of the

Km and Hyg selectable markers.

In conclusion, site-specific integration of a targeting cassette in the attP site with

concomitant removal of the residual selectable marker and stable transmission of the SSI

allele from the R2 to the R3 generation did occur in three of the nine analyzed transformants

(13-3, 16-11 and 16-12).

Molecular confirmation of germ line transmitted SSI events: PCR and Southern

blot analysis

Genomic DNA was isolated for PCR and Southern blot analysis from the Km-resistant

homozygous R3 or R4 (see Table 3) SSI offspring plants of the 16-11, 16-12 and 13-3

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transformants and from the progeny plants of the 16-4 transformant without an SSI

recombinant product. Amplification with primers a and b showed that all R1 16-11 derived

progenies (Table 3 (5-8)) harbor the recombination event I (Figure 1d), while in none of

these transformants the original attP-PCR fragment could be amplified. Interestingly, from

some of the R1 transformants, the two different recombination outcomes could be recovered.

The a/b primer set amplified the PCR fragment indicative for recombined outcome I in two

out of four analyzed 16-12 progenies and one out of three 13-3 progenies (Table 3

(9,10,15)), while the primer set a/c amplified the fragment diagnostic for recombined

outcome II in the other progenies (Table 3 (11-14)). Primer set a/d amplified the predicted

PCR product for the target attP site in all 16-4 progenies without an SSI event (Table 3 (1-4)).

The seven obtained outcome I PCR fragments with the a/b primer set were also sequenced

and the sequencing results confirmed precise recombinant attL products in the leaf DNA of

these transformants.

In parallel, we performed Southern blot analysis on the same DNA samples. DNA was

digested with either NcoI or SphI (Figure 2a-e). The blots with NcoI-digested DNA were

hybridized with the INT probe and the blots with SphI-digested DNA with the GUS, HPT,

NPTII and INT probe (Figure 1 and 2). The INT probe (probe 2) is predicted to detect a 2.6-

kb NcoI hybridizing fragment in the attP-containing target construct (Figure 1a), while it

should reveal either a 1.8-kb or a 2.8-kb NcoI fragment for recombined outcome I (Figure 1d)

or II (Figure 1e), respectively. The GUS probe should detect a 3.7-kb SphI hybridizing

fragment in plants with outcome I specific for the targeting construct (Figure 1d), while no

signal should be present in plants with recombined outcome II, and one or more T-DNA–

plant junction fragments bigger than 4.1 kb should be detected if random integration of the

targeting construct has occurred (Figure 1b). When using the HPT probe after SphI DNA

digestion, no hybridizing fragments should be detected in the recombinants, while one T-

DNA–plant junction fragment bigger than 2.2 kb should appear in the case of random

integration of the targeting construct (Figure 1a). Furthermore, the NPTII probe should detect

the same hybridizing T-DNA–plant junction fragment bigger than 1.8 kb in recombinants with

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outcome I and II (Figure 1d,e), while this T-DNA–plant junction fragment should be of a

different size in transformants with random integration of the targeting construct (Figure 1b).

Finally, the same T-DNA–plant junction fragment bigger than 3.3 kb should appear in all

SphI-digested DNA samples when using the INT probe (Figure 1a,d,e).

In agreement with the PCR analysis, Southern blot results confirmed the targeted

integration in 16-11, 16-12 and 13-3 homozygous offspring (Figure 2). Indeed, the expected

hybridization fragments for SSI outcome I and II were detected on the NcoI/INT (Figure 2a)

and SphI/GUS (Figure 2b) blots, indicating that in these lines SSI had occurred at the attP

target site as predicted from the PCR analysis. In the offspring of R1 transformants 16-12

and 13-3, both recombined outcomes I (Fig. 2a lanes 9, 10, and 15) and II (Fig. 2a lanes 11,

12, 13 and 14) were found. As predicted, in offspring with recombined outcome II, a 2.8-kb

NcoI fragment was detected with the INT probe and no 3.7-kb hybridization fragment with

the GUS probe, while the lines with recombined outcome I showed the expected 1.8-kb NcoI

band with the INT probe (Figure 2a) and the 3.7-kb SphI fragment with the GUS probe

(Figure 2b). On the contrary, in the offspring of the non-SSI R1 16-4 transformation event,

the internal attP-containing 2.6-kb NcoI hybridizing fragment was observed with the INT

probe (Figure 2a). In addition, in the R1 16-4 offspring, a new 4.8-kb SphI hybridizing

fragment was seen with the GUS probe, corresponding to a T-DNA–plant junction fragment

(Figure 2b).

An additional weak INT probe hybridizing NcoI fragment of about 5.5 kb was detected in

most of the SSI outcome I recombinants. Similarly, an additional GUS hybridizing SphI

fragment of about 8 kb was seen in all but one of the SSI outcome I recombinants

specifically (Figure 2b). In the R1 13-3 SSI offspring for both outcome I and II recombinants,

multiple extra hybridization bands were observed with the GUS probe probably

corresponding to the integration of additional copies of the targeting vector at the target

locus (Fig. 2b lanes 13,14,15).

Further, the absence of a HPT hybridizing fragment in the offspring of the R1 16-11 and

16-12 transformants and one R1 13-3 progeny plant as expected for recombinant outcome I

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was confirmed (Fig. 2c, lanes 5-12,15), while in R1 13-3 offspring plants with outcome II, a

HPT hybridizing fragment of about 5 kb was observed (Figure 3c, lanes 13-14). As the

segregation analysis of these 13-3 offspring plants on Hyg-containing medium demonstrated

100% sensitivity towards Hyg (Table 3), we attribute this to a truncated HPT hybridizing

fragment, most likely linked to the extra GUS hybridizing fragments, still present in this

complex SSI locus.

Also, as expected for both recombinant outcome I and II, the same NPTII hybridizing

plant–T-DNA left border fragment was observed. Indeed, an NPT probe hybridizing SphI

fragment of about 6.5 kb was observed in all SSI recombinants (Figure 2, lanes 5-14) except

in the complex R1 13-3 recombinant outcome I (Figure 2, lane 15). The R1 16-4 progeny

plants without an SSI event contained a different NPT hybridizing T-DNA–plant junction

fragment of 4.7 kb (Fig. 2d, lanes 1-4). Also, additional NPTII hybridizing fragments were

seen in the 16-12 recombinants with outcome II and in all the 13-3 recombinant progenies

with both outcomes. This can only be explained by rearrangements during the SSI event at

the left T-DNA end or by a complex targeting T-DNA integration pattern.

Finally, the expected T-DNA–plant junction fragment bigger than 3.3 kb was present in

all SphI-digested DNA samples after hybridization with the INT probe, confirming the single-

copy integration pattern of the target locus and no rearrangements at the right T-DNA plant

junction in transformants with or without an SSI event.

From this molecular data analysis, we can conclude that among the identified SSI

integration events, at least one, ie the progeny plant from R16-12 (Figure 2, lane 9) shows

the exactly predicted desired outcome, namely integration in the PP’ target site and removal

of the resident selectable marker with only one hybridizing fragment with all the probes and

digests tested. The other progenies with recombined outcome I (16-11, 16-12 and 13-3) do

show the correct recombinant PCR fragments, do not have the resident Hyg resistance

marker, show the expected GUS- and INT hybridizing restriction fragments, but do contain in

addition other fragments most likely due to aberrancies in the integration and resolvement

process.

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Determination of the presence of LB-loxP-RB footprints

Next, we determined whether the recombinants with a targeted T-DNA integration event

contained footprints of random integration. Indeed, if random T-DNA integration of the

targeting construct occurs and the sequence between the loxP sites is subsequently deleted

by CRE-mediated site-specific recombination, an LB-loxP-RB footprint will remain. To this

end, the primer combination e/f (Figure 1b) was used and was predicted to yield a PCR

fragment of 591 bp in case of CRE-mediated excision of the DNA between the loxP sites in

the correctly processed T-DNA from the targeting vector. For most of the homozygous

offspring lines of R1 16-11, 16-12 and 13-3 transformants amplification of this specific

fragment (Table 3) was obtained, indicating that LB-loxP-RB footprints were present. Only in

one of the R1 16-12 progenies this footprint segregated from the targeted SSI locus.

Furthermore, also the progenies from R1 transformants with random integration events of

the targeting construct (such as those from 16-4) contained the LB-loxP-RB footprint,

indicating that one or more targeting T-DNA insertions did occur from which the internal T-

DNA was deleted.

DISCUSSION

For the insertion of new DNA at a known locus, both recombinase-mediated and

homologous recombination-dependent site-specific DNA targeting have been demonstrated

to work. For homologous recombination (HR), targeting frequencies of 10-3-10-6 have been

reported in plants, being still quite inefficient (Britt and May, 2003; Hanin and Paszkowski,

2003; Iida and Terada, 2004). Studies have shown that double stranded breaks (DSBs)

induced by site-specific edonucleases can stimulate the HR repair machinery and thus gene

targeting (Puchta, 2005). Also, in planta gene targeting was achieved in Arabidopsis with a

frequency up to 8.3 x 10-3 by crossing an endonuclease expressing locus with a target and a

transgenic donor locus both containing the corresponding restriction sites in homologous

sequences, resulting in recombination between the activated target site and both ends of the

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released donor construct (Fauser et al., 2012). Recently, sequence-specific nucleases

introducing double-stranded breaks such as zinc-finger nucleases (Shukla et al., 2009;

Townsend et al., 2009; Tzfira et al., 2012), TALEN nucleases (Christian et al., 2010; Tzfira et

al., 2012; Zhang et al., 2013) and mega-nucleases (Yang et al., 2009; Gao et al., 2010;

Tzfira et al., 2012) were shown to all target DNA integration to those double-stranded breaks

with reproducible frequencies. On the other hand, site-specific recombination systems have

been described to allow gene targeting (Wang et al., 2011 and references therein).

Compared to homology dependent gene replacement, which involves end-joining ligation,

site-specific recombinases are precise, but need pre-existing recombination sites in the DNA.

Here, we demonstrate that the combined activities of the unidirectional φC31 integrase

and the reversible CRE recombinase can integrate a T-DNA at a predetermined position in

the Arabidopsis genome upon Agrobacterium-mediated floral dip transformation with

concomitant removal of the resident selectable marker. Indeed, one cycle of ISSI of an

Agrobacterium-delivered T-DNA is shown to have occurred by PCR, Southern blot, and

segregation analyses in the offspring of three out of 35 selected T-DNA transformants.

Fragments of the expected sizes were detected on Southern blots and the selectable marker

from the target locus was lost in the homozygous SSI recombinants. In addition, the

identified integration events with outcome I are Km-resistant, and contain the added GOI

(GFP/GUS) in the target locus.

From the results, it is clear that random insertion of the targeting T-DNA did happen in

most, if not all, of the transformed female gametocytes, for which an R1 Km resistant

seedling could be selected. First of all, most of the obtained R1 transformants lost the Km

selectable marker during the first generation. This is probably the result of CRE mediated

excision of random integrated T-DNA copies, generating a circular extra-chromosomal

product which then gets degraded or lost upon cell division. Indeed, the nine R1

transformants with a PCR proven SSI event in the leaves did not transmit the Km resistance

as predicted, and only few R2 seedlings were still Km resistant. Also, more than half of the

other 26 R1 Km-selected transformants without SSI-specific PCR fragments in the leaves

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showed loss of the Km resistance in the R2 generation. Furthermore, LB-loxP-RB footprints

were found in most of the transformants and also in the offspring of the three SSI site-

specific recombinants. This was also observed by Vergunst et al. (1998). These footprints

should anyway be removed from the final recombinant, which can be easily done by

segregation or through back-crosses, because the RB-loxP-LB footprints could possibly

interact with other loxP sites in the genome (SSI locus or others).

Altogether, the results suggest that the CRE recombinase activity was too low in the

transformed female gametocyte to efficiently obtain targeted integration. Indeed, it was

already observed before that the CRE activity was lower in germinal cells compared to

somatic cells when expressed from a 35S driven expression cassette (Marjanac et al.,

2008). Thus, to succeed optimally in the proposed ISSI set-up and to drive targeted

integration immediately after T-DNA transfer, one needs to have both the CRE recombinase

and the INT integrase simultaneously working well in the transformed female gametocyte. In

particular the CRE recombinase needs to be very active to generate the circular T-DNA

product and to prevent random insertion. Therefore, adequate female gametocyte specific

promoters could be used as described by Van Ex et al. (2009).

Further, analysis of the R2 and R3/R4 progenies of selected R1 transformants with an

SSI event in the leaves led to the following insights. The randomly integrated targeting T-

DNA construct in R2 plants could serve as donor locus for release of CRE-mediated excised

circles which were then either inserted stably in the attP target locus or got lost. Probably

site-specific integration did occur independently in many different somatic cells. This

explains why several of the R1 transformants with an SSI-specific PCR fragment amplified

from R1 leaf material, did not transmit this SSI event to the R2 and R3 generation.

Srivastava and Ow (2003) described the rare instance of the maintenance of the CRE-

mediated deletion product as an extra-chromosomal circular molecule in mature somatic

tissue. In our experiments the CRE gene was expressed by the constitutive 35S promotor,

which is active at all stages of development. Somatic accumulation of CRE-mediated

excision circles could also have occurred in our experiments yielding donor molecules for

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SSI into the target locus of somatic cells. Also Arumugam et al. (2007) found that CRE-

mediated excisions occurred with very high efficiency in quiescent, differentiated somatic

tissues, but were less abundant in meristematic tissues.

Only if the SSI occurs in the primordial cells of the germ line it is stable and transferred

to the next generation. This can occur several times during development. Indeed, several R2

seedlings selected to still contain the Km resistant targeting construct, acquired independent

SSI events during development of the primordial germ cells, as both SSI recombined

outcome I and II events were found in the R3 generation. We postulate that the excised

targeting construct recombined in different attP sites in different germline primordial cells,

resulting in offspring with two different SSI alleles.

Finally, in some of the selected R1 transformants, the randomly integrated targeting T-

DNA remained stably present in the genome, and CRE-mediated loss of the Km resistant

marker did not occur. This can be explained by either the absence of one of the loxP sites

due to truncation of the T-DNA ends, or to mutated or inaccessible loxP sites. Previous

reports also documented that CRE-mediated excision can be suppressed in a significant

amount of transformants because of deleted, mutated or non-accessible loxP sequences

due to chromatin modifications in complex loci (De Buck et al., 2007; Marjanac et al., 2008;

De Paepe et al., 2009).

For certain applications, one would eventually like to obtain CRE- and INT-free plants

containing the stacked insert locus. The most straightforward approach is to flank the CRE

and INT expression cassettes with directly oriented site-specific recombination sites other

than loxP sites (f.e. FRT or RS sites), so that subsequent introduction of the corresponding

recombinase by co-transformation, crossing or re-transformation would excise the CRE and

INT gene expression cassettes. Moreover, the selectable marker could also be excised in

this manner to obtain a final marker-free T-DNA integration event. Also, the incorporation of

a promoter-trap target construct, where the loxP site is placed between the promoter and the

coding region of the selectable marker in the target construct, and the use of a targeting

vector with a loxP site at the promoterless 5’ end of the coding region of a second selectable

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marker (Dafhnis-Calas et al., 2005; Wang et al., 2011 and references therein) may aid in

obtaining only precise SSI lines because then transformants with random T-DNA events will

not survive on selective growth medium. Further, optimization of the promoters of both

recombinases could greatly enhance the targeting frequency at the moment of the

transformation/integration event itself. In addition, additional single-copy target lines could be

compared in a follow-up study to select the best performing target line and to obtain a clear

view of the effciency of the proposed SSI method.

The ISSI described here opens new ways for the development of transgenics with

single-copy and stable multigene inserts at one genetic locus. The SSI integrated

recombined product (recombined outcome I) can now be the target for stacking another

sequence by site-specific recombination (Figure S2) and the ISSI cycle can be repeated for

an arbitrary number of times with only two selectable markers and two sets of recombination

sites, namely the loxP and the attB/attP sites. This is in contrast to the stacking possibilities

of the recombinase-mediated DNA cassette exchange (RMCE) method (Li et al., 2009)

which is dependent on the availability of compatible recognition sites. The possibility to stack

multiple transgenes at a single locus should be very useful in the near future. A merit of

assembling transgene arrays is that it will avoid the difficulties arising from segregation of

independent transgenic loci. In addition, stacking of multiple transgenes at a predicted site of

integration ensures single copy insertions and allows control of the position in the genome.

EXPERIMENTAL PROCEDURES

Plasmid construction: pHsc1, pCre and pK2L2B vectors

To produce the targeting construct pK2L2B, compatible oligonucleotides (5’-

GGCCCGTTAACATAACTTCGTATAGCAT-3’ and 5’-ACATTATACGAAGTTATCCATATTA-

3’) containing the loxP sequence were first ligated and inserted into the PspOMI/BstXI site of

pXD610 (De Loose et al., 1995). Then, compatible oligonucleotides (5’-

AATTCAGATCTATAACTTCGTATA-3’ and 5’-ATGTATGCTATACGAAGTTATG-3’)

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containing the second loxP sequence in the same orientation were ligated and inserted into

the EcoRI/BamHI site. Next, compatible oligonucleotides (5’-

CGTTAACCGGTGCGGGTGCCAGGGCGTGCCC-3’ and 5’-

TTGGGCTCCCCGGGCGCGTACTCCACTCTAGAGAGCT-3’) containing the attB sequence

were ligated and inserted into the SacI/HpaI site of the p35S-Fs7 vector, which contains the

P35S::eGFPGUS expression cassette, followed by insertion of compatible oligonucleotides

(5’-GGCCCTCTAGACGGTGCGGGTGCCAGGGCGTGCCC-3’ and 5’-

TTGGGCTCCCCGGGCGCGTACTCCACCATATG-3’) containing the second attB site into

the PspOMI site. Finally, the XbaI-cut attB-GUS/GFP-attB fragment was ligated to the XbaI-

cut loxP-containing fragment to yield the pK2L2B vector. Plasmid pHsc1, which harbors the

φC31 integrase coding cassette (De Paepe et al., 2009), and the CRE T-DNA were

described previously (De Buck et al., 2000; Marjanac et al., 2008; De Paepe et al., 2009).

Agrobacterium strain, plant transformation and selection of transformants

The pHsc1 and pK2L2B plasmids were transferred to the Agrobacterium tumefaciens strain

C58C1RifR(pMP90) by electroporation. Homozygous single-copy Cre1 (in C24 background)

(Marjanac et al., 2008) plants were transformed with the pHsc1 vector by the floral-dip

method (Clough and Bent, 1998). Seeds of the dipped plants were harvested and sown on

germination medium supplemented with Hyg (20 mg/L) for selection of primary

transformants (T1). A single-copy transformant, Cre-Hsc1, was chosen as target for

supertransformation. T2 progeny plants of the Cre-Hsc1 plant (homozygous or hemizygous

for the Hsc1 locus), the homozygous Cre1 plant (Marjanac et al., 2008) and C24 wild-type

plants were transformed with the pK2L2B T-DNA vector. Selection of the R1 seedling

transformants was performed on Km (50 mg/L). After growing and selfing these plants, R2

seed stocks were obtained and the zygosity of the Km locus and the presence of Hyg

resistance was determined in the R2 and R3 progeny by sowing at least 50 seeds on either

Km- or Hyg-containing medium (20 mg/L).

Plant DNA preparation and Southern blot analysis

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DNA of Arabidopsis leaf material was prepared from 200 mg frozen plant tissue using the

DNeasy Plant Mini Kit as described by the manufacturer (Qiagen, http://www.qiagen.com/).

Digested Arabidopsis DNA (1 µg) was loaded into each lane of a 1% agarose gel. For the

Hsc1 T-DNA, the HPT (Figure 1a, probe 1) and the INT (Figure 1a, probe 2) probes were

used. The HPT and INT probes were prepared via PCR amplification from the plasmids

pH610 (De Buck et al., 1998) and pGEMINT (De Paepe et al., 2009), respectively, with the

following primers: for the HPT probe with hpt1 (5’- ACCTGCCTGAAACCGAACTGC-3’) and

hpt2 (5’-GCGTCTGCTGCTCCATACAA-3’) and for the INT probe with anpae32 (5’-

ACGCCTGAAGCTCATACCAC-3’) and anpae33 (5’- GGAAACGTCATGGACCTGAT-3’). For

the K2L2B T-DNA, the NPTII (Figure 1b, probe 3) and the GUS (Figure 1b, probe 4) probes

were used. The NPTII probe was PCR amplified from the pK2L610 plasmid (De Buck et al.,

1998) with the primers napod8 (5’- GCTTTTCTGGATTCATCGACTGTG-3’) and napod13

(5’- CGCTATAAACCTATTCAGCACA-3’), and the GUS probe was amplified from the

pGEMgus plasmid (De Buck et al., 1999) with the primers GUS3F (5’-

TGGCAGTGAAGGGCCAACAG-3’) and GUS5R (5’- TACCTGTTCACCGACGACGG-3’).

The probe DNA was labeled and detected using the PCR DIG probe synthesis mix (Roche,

Mannheim, Germany), Anti-digoxigenin (DIG)-AP Fab fragments and the CDP-star detection

module (Roche) (De Paepe et al., 2012).

PCR analysis

For PCR analysis, 100 ng of leaf DNA was used. Primers were: a, 5’-

CGCCTGGTTCTTCTTTTTCA-3’; b, 5’-TAACCATCTGTGGGTCAGCA-3’; c, 5’-

TCAGTGACAACGTCGAGCACA-3’; d, 5’-CGGAATTGACGGATCTGGTA-3’; e, 5’-

CAGGCCAAATTCGCTCTTAG-3’; f, 5’- CCTGGCCTAACATCACACCT-3’. The PCR

reaction conditions were: initial denaturation at 94°C for 5 min, followed by 30 cycles of

denaturation at 94°C for 45 sec, annealing at 56°C for 45 sec, and elongation at 72°C for 60

sec, with a final elongation step at 72°C for 5 min. 10 µL of the PCR samples was loaded on

1% agarose gels and visualized by addition of EtBr.

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ACKNOWLEDGEMENTS

The authors thank William Brown (Institute of Genetics, University of Nottingham, UK) for

providing the plasmid with the φC31= integrase gene, our colleagues of the floral dip and

sequencing groups, Marta Walczak for help with experimental work, and Annick Bleys for

help in preparing the manuscript. This work was supported by grants from the 6th

Framework Program of the European Union ‘GENINTEG’ (LSHG-CT2003-503303), the

European Union BIOTECH program (QLRT-2000-00078), with additional co-financing from

the Flemish Community and the Research Foundation Flanders (G.021106).

CONFLICTS OF INTEREST

No conflicts of interest are to be declared.

SUPPORTING INFORMATION

Additional Supporting Information may be found in the online version of this article:

Figure S1. Schematic outline of the alternative ISSI process when using the pK2L2B vector

for site-specific recombination into the Cre-Hsc1 target line.

Figure S2. ISSI T-DNA stacking strategy.

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Tables

Table 1: Transformation efficiency of the targeting T-DNA construct and PCR analysis of R1

transformants for site-specific recombination

Recipient T2

Cre-Hsc1

planta

# of sown

seeds/

plant line

# of

Kmr R1

transfor-

mants

Transformation/

recombination

efficiency (TE)

(%)b

Positive R1

PCR for

recombined

outcome Ic

Positive R1

PCR for

recombined

outcome IId

R1 plant with

positive SSI

PCR product

4 2000 7 0.35 0 2 4-1

4-2

7 2000 4 0.2 0 1 7-1

13 2000 3 0.15 0 2 13-2

13-3

15 2000 6 0.3 0 0

16 4000 12 0.3 3 1

16-3

16-4

16-11

16-12

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19 1000 0 0 0 0

23 1000 3 0.3 0 0

Total 14000 35 0.25 3 6

Cre1 4000 5 0.13 0 0

C24 4000 18 0.45 0 0

Kmr, kanamycin resistant

a Seven T2 progeny plants, Ho or He for the Hsc1 locus (Hyg resistant), from a single T1

transformant being Ho for the CRE locus, were numbered and used for floral dipping with the

pK2L2B construct.

bTransformation/recombination efficiency: the # of transformants divided by #

seeds/plant*100.

c PCR reaction with primers a and b yields the 478-bp fragment if the recombined outcome I

is present in leaf DNA of the R1 plant (see Figure 1d).

d PCR reaction with primers a and c yields the 1022-bp fragment if the recombined outcome

II is present in leaf DNA of the R1 plant (see Figure 1e).

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Table 2: Segregation analysis of the targeting construct containing the Kmr marker and the

target locus containing the Hygr marker in the nine selfed progenies of R1 transformants

positive for the SSI-specific PCR

tested R2 #Kmr/total #Hygr/total tested R3 #Kmr/total #Hygr/total

seed stock R2 R2 seed stock R3 R3

4-1 5/60 58/58 4-1-1 34/50 50/50

4-1-2 37/50 50/50

4-1-3 34/50 50/50

4-1-4 40/52 50/50

4-2 26/60 49/60 4-2-1 46/60 0/58

4-2-2 42/57 55/55

4-2-3 45/60 54/54

4-2-4 40/58 33/57

4-2-5 42/57 51/51

7-1 48/60 46/60 7-1-1 40/59 58/58

7-1-2 41/60 60/60

13-2 0/58 46/60

13-3 30/60 9/60 13-3-1 49/57 0/58

13-3-2 41/59 0/59

13-3-3 47/59 0/58

13-3-6 46/59 0/60

13-3-7 46/60 0/59

16-3 36/58 59/59 16-3-1 39/55 58/58

16-3-2 28/52 58/58

16-3-3 35/56 58/58

16-3-4 37/50 58/58

16-3-5 23/54 58/58

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16-4 5/60 60/60 16-4-1 44/56 58/58

16-4-2 50/50 52/52

16-4-3 58/58 58/58

16-11 3/78 60/60 16-11-1 42/54 33/53

16-11-2 58/58 0/58

16-11-3 58/58 0/58

16-12 2/60 59/59 16-12-1 21/59* 38/58

16-12-2 7/60* 38/56

Kmr, kanamycin resistant; Hygr, hygromycin resistant

ISSI positive lines are underlined: if the site-specific integrants were derived from a

hemizygous target plant, the R2 plant should be all Hyg-sensitive and all derived R3 seeds

should be sensitive to Hyg while segregating (3:1) on Km or 100% resistant to Km. If the

site-specific recombinants were derived from a line homozygous for the Hsc1 locus, the R3

seeds should segregate on Hyg and Km or should yield progeny for 100% sensitive to Hyg

and 100% resistant to Km; progeny plants of the R3 or R4 seed stocks indicated in bold

were tested by PCR (see Table 3).

* We observed less than expected Kmr seedlings in the 16-12 R3 progeny seed stocks.

However in the next R4 generation, normal segregation patterns for a single locus insert

were obtained.

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Table 3: PCR analysis on leaf DNA of SSI and non-SSI transformants homozygous for the

targeting Kmr construct.

(e) Plant Generation Positive PCR for

target locus

(attP site)a

Positive PCR for

recombined

outcome Ib

Positive PCR for

recombined

outcome IIc

Positive PCR

LB-RB footprintd

Non-SSI

1 16-4-3-4 R3 + - - -

2 16-4-2-5 R3 + - - +

3 16-4-2-4 R3 + - - +

4 16-4-2-3 R3 + - - +

SSI

5 16-11-2-1 R3 - + - +

6 16-11-2-3 R3 - + - +

7 16-11-3-8 R3 - + - +

8 16-11-1-2 R3 - + - +

9 16-12-1-2-9 R4 - + - -

10 16-12-1-2-8 R4 - + - +

11 16-12-2-3-7 R4 - - + +

12 16-12-2-3-10 R4 - - + +

13 13-3-7-1 R3 - - + +

14 13-3-6-1 R3 - - + +

15 13-3-1-2 R3 - + - +

a Primers a and d yield the 305-bp fragment present in the original target locus.

b Primers a and b yield the 478-bp fragment diagnostic for SSI recombined outcome I.

c Primers a and c yield the 1022-bp fragment diagnostic for SSI recombined outcome II.

d Primers e and f yield the 591-bp fragment.

e The numbers in the first column are corresponding to the lanes used for Southern blot

analysis (see Figure 2).

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Figure legends

Figure 1 Schematic outline of the site-specific integration strategy when using the pK2L2B

vector for site-specific recombination into the Cre-Hsc1 target line. Sizes of the expected

fragments after digestion with SphI or NcoI when using the HPT (probe 1), the INT (probe 2),

the NPTII (probe 3) and the GUS (probe 4) probes are indicated. Primer pairs are shown as

a, b, c, d, e, f above the T-DNA construct schemes. (a) Structure of the T-DNAs in the CRE

and INT-expressing target line with an attP site (PP’) for targeted integration. The two T-

DNAs are inserted at different loci. The Cre locus is homozygous and is stably present. (b)

Structure of the targeting T-DNA vector with two copies of an attB site, the Km resistance

selectable marker, the P35S::eGFP/GUS expression cassette and two directly oriented loxP

sites next to the T-DNA borders. (c1) Structure of the CRE intermediate, obtained by CRE-

mediated circularization of the K2L2B T-DNA. (c2) and (c3) Structure of the INT

intermediates I (c2) and II (c3): INT activity will lead to site-specific recombination of the PP’

site in the target locus with attB I or attB II sites of the CRE intermediate. (d) Recombined

outcome I as a result of CRE activity on the INT intermediate I. (e) Recombined outcome II

as a result of CRE activity on the INT intermediate II. Abbreviations: LB, left border; RB, right

border; 3’ocs, 3’ end of the octopine synthase gene; NPTII, neomycin phosphotransferase II

gene; Pn, promoter of the nopaline synthase gene; T35S, 3’ terminator of CaMV; eGFPGUS,

eGFPGUS fusion gene of the enhanced green fluorescent protein and β-glucuronidase;

P35S, CaMV 35S promoter; 3’n, 3’ end of nopaline synthase gene; HPT, hygromycin

phosphotransferase gene; Pcas, cassava vein mosaic virus promoter (Verdaguer et al.,

1998); INT, integrase gene of bacteriophage φC31 (Dafhnis-Calas et al., 2005); 3’chs’, 3’

end of the chalcone synthase gene; CRE, CRE recombinase gene; BAR, phosphinothricin

transferase gene; PSSUARA, the small subunit promoter of Arabidopsis; TP, transit peptide;

3”g7, 3’ terminator of gene 7; SI, SphI; NI, NcoI. The open triangle represents the loxP site;

the open boxes with PP’ and BB’ symbolize the attP and attB recombination substrate sites

for the INT recombinase and the open boxes with BP’ and PB’ symbolize the attL and attR

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recombination sites. Note: the P35S::eGFP/GUS expression cassette can be replaced by

any desired DNA fragment.

Figure 2 Southern blot analysis on the progenies of transformants with site-specific

integration and a randomly inserted pK2L2B T-DNA integration. (a) DNA gel-blot of NcoI-

digested DNA hybridized with the INT probe. The target locus in the Cre-Hsc1 plant yields a

2.6-kb NcoI fragment and the expected NcoI fragment in the site-specific integrants is 1.8 kb

for recombined outcome I and 2.8 kb for recombined outcome II. (b) DNA gel-blot SphI

fragments detected using the GUS probe. The expected fragment in the site-specific

integrants is 3.7 kb for recombined outcome I. No hybridization bands are expected in

recombinants with outcome II. Random integration of the targeting construct will yield a right

T-DNA–plant junction of more than 4.1 kb. (c) DNA gel-blot of SphI-digested DNA hybridized

with the HPT probe. No hybridizing fragments should be detected in the recombinants, while

one T-DNA–plant junction fragment bigger than 2.2 kb is expected in the case of random

integration of the targeting construct. (d) DNA gel-blot SphI fragments detected using the

NPTII probe. The NPTII probe should detect the same hybridizing T-DNA–plant junction

fragment bigger than 1.8 kb in recombinants with outcome I and II, while this T-DNA–plant

junction fragment should be of a different size in transformants with random integration of

the targeting construct. (e) DNA gel-blot of SphI-digested DNA hybridized with the INT probe.

A T-DNA–plant junction fragment bigger than 3.3 kb is expected in all DNA samples. Lanes

1-4 contain DNA from different homozygous progenies of 16-4, lanes 5-8 that from 16-11,

lanes 9-12 that from 16-12 and lanes 13-15 that from 13-3. The numbers indicating the lanes

are also used in Table 3.

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