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First published online May 8, 2009; 10.1104/pp.109.137612 Plant Physiology 151:1087-1095 (2009) © 2009 American Society of Plant Biologists OPEN ACCESS ARTICLE
Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange[OA]DuPont/Pioneer Crop Genetics, Experimental Station, Wilmington, Delaware 19880
A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.
Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005  ; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.
Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002
Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995
When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000
RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005 To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization.
Design of FLP-FRT-Mediated RMCE
The target QC288A and donor QC329 DNA each contained a FRT1 site and a FRT87 site in the same orientation (Fig. 1, A and B
; Tao et al., 2007
The QC288A DNA contained a selection gene, hpt, driven by the constitutive promoter scp1, and transgenic events were selected with hygromycin. The QC329 DNA contained a promoterless selection gene, als, that would not be expressed unless a promoter was placed upstream of it. FLP-mediated DNA recombination could bring the promoterless als gene of QC329 downstream of the scp1 promoter of QC288A to form QC288A329 to enable retransformation events being selected with chlorsulfuron. The random integration events of QC329 would not survive the selection unless the promoterless als gene inserted by chance downstream of a native promoter. The fluorescent protein genes yfp in QC288A and cfp in QC329 and QC288A329 were used to screen transgenic events (Fig. 1, A, B, and D).
A total of 82 target events were produced with QC288A DNA. Somatic embryo samples of the events were analyzed by quantitative PCR (qPCR) specific to the scp1, hpt, and yfp genes, regular PCR specific to the 5' end, 3' end, and full-length QC288A, and Southern hybridization with hpt and yfp probes to identify single complete-copy events. A total of 33 such events regenerated into T0 plants, and their leaf samples were analyzed by similar qPCR, PCR, and Southern analyses. Sixteen seeds from one self-pollinated T0 plant of each of 10 selected events (lines) were planted to get T1 plants, which were then analyzed by scp1-, ubiq10-, and yfp-specific qPCR assays to check for segregation. Eight lines produced homozygous T1 plants. Three lines, A, B, and C, were used in this report (Table I ).
Genomic DNA fragments bordering the QC288A transgene in six target lines were cloned and sequenced. Genomic DNA sequences 601, 984, and 496 bp bordering the 5' ends of lines A, B, and C, respectively, and sequences 2,588, 1,305, and 543 bp bordering their 3' ends were obtained. The alignment of the border sequences with the QC288A sequence revealed that line A lost 5 bp from the 5' end and none from the 3' end, line B lost 17 bp from the 5' end and 49 bp from the 3' end, while line C lost 22 bp from the 5' end and 11 bp from the 3' end. None of the transgene end losses was long enough to affect the FRT recognition sites. BLASTN search of the border sequences did not yield any significant homology with any known sequences in the National Center for Biotechnology Information database (www.ncbi.nlm.nih.gov).
Suspension cultures initiated from the developing embryos of target lines A, B, and C homozygous T1 plants were retransformed with the donor DNA QC329 cobombarded with the flp DNA QC292. Three putative retransformation events resistant to chlorsulfuron from target A, six from target B, and three from target C were screened at the callus stage for the reporter gene cfp expression followed by a common PCR to check DNA recombination at the FRT1 site (Table II ). Events B5 and B6 were derived from the retransformation of the original hemizygous target B callus that had never gone through plant regeneration. All events were then evaluated by four construct-specific qPCR analyses (Fig. 1) to check for DNA recombination at the FRT1 site and the presence of the target, donor, and flp DNA (Table II), followed by five border-specific PCR analyses specific to each target line using the 5' border, 3' border, and transgene-specific primers (Fig. 1, A, D, and E) to confirm DNA recombination at and between the FRT1 and FRT87 sites (Fig. 2 ).
For example, event A1 was positive for both CFP expression and the FRT1 site-specific PCR. The construct-specific qPCR analyses revealed that event A1 had one copy of RMCE, contained two copies of the donor, and was free of either the target or flp (Table II). The border-specific PCR analyses revealed that event A1 was positive for both the 5' end and 3' end assays specific to RMCE (Fig. 2, A and B), negative for either the 5' end or the 3' end assays specific to the target (Fig. 2, C and D), and positive for a small excision-specific band amplified by the full-length PCR (Fig. 2E). Since one copy of RMCE was simultaneously detected with the excision in the homozygous target-derived event A1, the event had to be an RMCE-excision, with one target converted to RMCE and the other converted to excision. The expected large RMCE-specific band (6,652 bp) of event A1 failed to be amplified by the same full-length PCR due to its competitive disadvantage to the small excision-specific band (1,307 bp). Based on similar qPCR (Table II) and border-specific PCR analyses (Fig. 2), event A2 was an RMCE-excision event containing a copy of the donor and flp DNA. Event A3 was a homozygous target escape containing five copies of the donor. The target-specific band of A3 was amplified by the full-length PCR (Fig. 2E). Events B1, B2, and B4 were clean RMCE-excision events containing no additional donor or flp DNA insertions. Event B3 was an RMCE-excision containing a donor. Event B5 was an RMCE-wt (for wild type) event, since it was derived from the retransformation of the original hemizygous target B callus. Accordingly, no excision band was detected in event B5 by the full-length PCR even though the large RMCE-specific band was amplified (Fig. 2E). The 1.0 copy of the target in event B5 probably was a partial copy, since the target border-specific PCR did not detect it (Fig. 2, C and D). Event B6 was a hemizygous target escape, also containing the partial target, since 1.8 copies of the target were detected. The detection of the partial target in events B5 and B6 suggested that the original target B callus was chimeric. Event C1 was an RMCE-excision event with some cells still containing the target detected as 0.01 copy, which was confirmed by the border-specific PCR (Fig. 2, C and D). Events C2 and C3 were homozygous RMCE-RMCE events containing two copies of RMCE and one copy of the donor. Accordingly, the border-specific PCR failed to detect any target- or excision-specific bands but amplified the large RMCE-specific band (Fig. 2, C–E). To summarize, two, five, and three RMCE events were obtained from the retransformation of five, five, and six plates of target A, B, and C cultures, respectively, and the RMCE retransformation frequencies were thus calculated as 0.4, 1, or 0.5 event per plate (Table II). The average of these frequencies is approximately 10 times lower than the average five events per plate frequency for standard soybean biolistic transformation but high enough for routine RMCE event production.
Viable T0 plants regenerated from events A2, B5, C2, and C3 were analyzed by the same construct-specific qPCR analyses (Table II). Four A2 plants, A2-1, A2-2, A2-3, and A2-4, three C2 plants, C2-1, C2-2, and C2-3, and two C3 plants, C3-1 and C3-2, all retained the same molecular signatures of their respective callus parents A2, C2, and C3. The 1.0 copy of target detected in B5 callus was no longer observed in T0 plants B5-1, B5-2, and B5-3. The same border-specific PCR analyses also confirmed that the T0 plants were the same as their respective callus parents (Fig. 3 ).
Since the target QC288A and the RMCE QC288A329 sequences diverge downstream of the FRT1 site with hpt in QC288A and als in QC288A329 and upstream of the FRT87 site with yfp:nos in QC288A and cfp:nos in QC288A329 (Fig. 1, A and D), the alignment of the target and RMCE transgene sequences with their map sequences should confirm RMCE at the sequence level. The 30 bands, marked "x" in Figure 3, amplified from the target samples A, B, and C, RMCE callus samples A2, B5, and C2, and representative RMCE T0 plant samples A2-1, B5-3, C2-1, and C3-1, were cloned and sequenced. The alignment of the transgene sequences with predicted target, RMCE, and excision sequences confirmed accurate DNA recombination for all of the RMCE and excision events (data not shown). The sequences of the A2 and A2-1 excision-specific fragments (Fig. 3E) matched a predicted excision sequence containing an FRT1 site.
T0 plants A2-1, A2-2, A2-3, A2-4, B5-1, B5-2, C3-1, and C3-2 produced seeds. T1 plants germinated from these seeds were analyzed by the same construct-specific qPCR analyses. Since the T0 plants of each A2, B5, or C3 event were identical (Table II), T1 plants from different T0 plants of the same event were treated as one population for segregation analysis. The RMCE-specific qPCR would detect two copies, one copy, or zero copies of RMCE for RMCE-RMCE, RMCE-excision, and excision-excision. The target, donor, and flp-specific qPCR would detect two copies, one copy, or zero copies of the respective genes for homozygous, hemizygous, or null target, donor, and flp. Since the A2 T0 plants were hemizygous RMCE-excision containing a donor and a flp (Table II), the excision would segregate away from RMCE and the donor and flp would segregate independently if they were not linked to the RMCE-excision locus. The RMCE-excision locus of 42 A2 T1 plants segregated as 12 RMCE-RMCE, 18 RMCE-excision, and 12 excision-excision. The donor and flp segregated together but independently from the RMCE as 15 homozygous, 16 hemizygous, and 11 null. One plant was RMCE-RMCE and seven plants were RMCE-excision, all free of any target, donor, or flp DNA. Since the B5 T0 plants, already free of any target, donor, or flp, were derived from a hemizygous target, they were hemizygous RMCE-wt (Table II). The 36 B5 T1 plants segregated as 11 RMCE-RMCE, 17 RMCE-wt, and eight wild type. Since the C3 T0 plants were already homozygous RMCE-RMCE but contained a donor (Table II), all 48 C3 T1 plants remained as RMCE-RMCE and the donor segregated independently as 12 homozygous, 24 hemizygous, and 12 null. Target homozygous plants A, B, and C, RMCE T0 plants A2-3, A2-4, B5-1, B5-2, C3-1, and C3-2, RMCE-excision T1 plants A2-3-1 and A2-3-2, excision-excision T1 plants A2-3-3 and A2-3-4, and RMCE-RMCE T1 plants B5-1-1, B5-2-1, C3-1-1, and C3-1-2 were selected for Southern hybridization analysis. NdeI digestion and hpt, scp1, ubq, and flp probes were used. NdeI cuts QC288A only once at position 1,119 and has to cut another NdeI site in the genomic DNA bordering the 5' end of the QC288A transgene to produce a fragment, consisting of the 5' end 1,119 bp of QC288A and the genomic DNA segment (Fig. 1A), that would hybridize to both the hpt and scp1 probes (Fig. 4, A and B ). NdeI cuts QC288A329 only once at position 4,395 and also the same NdeI site in the 5' genomic DNA border to produce a fragment, consisting of the 5' end 4,395 bp of QC288A329 and the same genomic DNA segment (Fig. 1D), that would hybridize to both the scp1 and ubq probes (Fig. 4, B and C). Thus, the RMCE QC288A329-specific band would be 4,395 – 1,119 = 3,276 bp larger than the corresponding target QC288A-specific band. The ubq probe was derived from a soybean endogenous gene and hybridized to an approximately 6-kb wild-type band. Additional ubq-only bands were specific to randomly integrated donor QC329 that contained only one NdeI site at position 3,715 (Fig. 1B). scp1-only bands were specific to excision that contained only the scp1 promoter. The excision-specific fragment was produced by digestion at the same NdeI site in the 5' genomic DNA border and another NdeI site in the 3' genomic DNA border (Fig. 1E). NdeI cuts the flp DNA QC292 only once at position 4,039 (Fig. 1C). Bands hybridized to both the flp and scp1 probes were specific to randomly integrated QC292 (Fig. 4, B and D).
The Southern hybridization results were consistent with previous qPCR and PCR results except for a large scp1 band detected in C3-1 and C3-2 and extra ubq bands detected in target A-derived plants (Fig. 4, B and C). The large scp1 band of C3-1 and C3-2 also hybridized to the ubq probe and disappeared with the donor in C3-1-1 and C3-1-2, and was considered a scp1 promoter mingled with the donor at an unlinked random insertion site. Four of the five ubq-only bands below the approximately 6-kb wild-type band detected in A2-3 and A2-4 (Fig. 4D) were likely partial copies of the donor, since qPCR detected only one donor insert (Table II). One of the ubq-only bands, not detected by the donor-specific qPCR, remained in RMCE-excision plants A2-3-1 and A2-3-2, and two remained in excision-excision plants A2-3-3 and A2-3-4.
Single-site SSI creates two directly oriented recognition sites vulnerable to excision that makes the recombination events unstable. Mutant lox sites (Albert et al., 1995 ; Srivastava and Ow, 2001), cre gene displacement, and transient cre expression (Albert et al., 1995; Vergunst et al., 1998) can be used to prevent the excision. A donor DNA can be circularized prior to integration by a recombinase to remove any unwanted components such as the vector backbone to prevent them from being integrated (Vergunst et al., 1998; Srivastava and Ow, 2001). To achieve marker-free SSI, a two-step approach was proposed to combine gene integration using one recombinase system such as Cre-lox with gene excision using another system such as FLP-FRT conditionally controlled by an inducible promoter (Srivastava and Ow, 2004).
RMCE using two recognition sites provides a flexible way for gene targeting. If two identical sites are used, they must be in opposite orientations to prevent excision, although the DNA segment between the two sites can flip (Nanto et al., 2005 When using a homozygous target, an RMCE event can be chimeric, with the target on one chromosome being converted to an RMCE and the other target on the homologous chromosome being converted to an excision or unchanged. The frequent occurrence of RMCE-excision and the lack of RMCE-target in our experiments indicate that the FRT1 and FRT87 sites are not completely incompatible and that the FLP-mediated DNA recombination is highly effective. RMCE can even occur simultaneously on two homologous chromosomes, as in the case of events C2 and C3. More likely, a homozygous RMCE has to be obtained at the T1 generation via segregation. Any donor or flp DNA integrated randomly at a separate genomic site in an RMCE event can be removed by segregation. The effective RMCE described here opens new ways for transgenic product development and transgene expression studies. Large DNA fragments can be integrated via RMCE, which seems to rely only on FLP-catalyzed interactions between FRT sites. Various target lines can be produced in advance and maintained as production lines to accept genes with various preferences for gene silencing, tissue-specific expression, agronomic performance, etc. Multiple genes can be stacked reversibly at the same genetic locus by multiple rounds of RMCE using different recombination sites.
DNA Construction
The target construct QC288 containing scp1-FRT1:hpt:nos+ubiq10:yfp:nos-FRT87, the donor construct QC329 containing FRT1-als:pinII+ubq:cfp:nos-FRT87, and the FLP expression construct QC292 containing scp1:flp:pinII were made following standard molecular cloning procedures through multiple steps using components from existing DNA constructs (Li et al., 2007
The target DNA scp1-FRT1:hpt:nos+ubiq10:yfp:nos-FRT87 was released as a 4,544-bp fragment QC288A with AscI digestion from QC288, resolved by agarose gel electrophoresis, and purified using a gel extraction kit (Qiagen). Soybean (Glycine max) embryogenic cultures were transformed with QC288A following the biolistic transformation protocol using 30 µg mL–1 hygromycin for selection (Li et al., 2007
Soybean genomic DNA was prepared from somatic embryo or leaf samples and analyzed by Southern hybridization with digoxigenin-labeled probes (Li et al., 2007
Genomic DNA bordering the QC288A transgene was acquired using the GenomeWalker kit (Clontech). Genomic DNA digested with EcoRV, DraI, HpaI, or StuI was ligated to adaptors and amplified by two rounds of PCR. The first PCR used adaptor-specific primer AP1 (5'-GTAATACGACTCACTATAGGGCACG-3') and QC288A-specific primers Scp1-A (5'-CTACTGTCCTTTTGATGAAGTGACAG-3') for the 5' end border and Vec-S1 (5'-GATCGGGAATTCTAGTGGCCGG-3') for the 3' border. The second PCR used adaptor-specific primer AP2 (5'-CTATAGGGCACGCGTGGTCGAC-3') and QC288A-specific primers Scp1-A4 (5'-CTGGGCAATGGAATCCGAGGAG-3') for the 5' end border and Vec-S2 (5'-GCTGATGATCCCGGTGAAGTTCC-3') for the 3' border. Specific PCR fragments were cloned in pCR2.1-TOPO vector with the TOPO TA cloning kit (Invitrogen). Plasmid DNA was prepared with the Qiaprep plasmid DNA kit (Qiagen) and sequenced using a capillary DNA analyzer and the dye terminator cycle DNA sequencing kit (Applied Biosystems). Sequence assembly and alignment were done using Vector NTI programs (Invitrogen). Sequence searches were done using the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) advanced BLAST algorithm.
PCR was done on leaf or somatic embryo DNA samples following the same protocol (Li et al., 2007 Putative RMCE events were screened by CFP expression with a MZFLIII stereo microscope (Leica Microsystems) and identified by PCR with primers 35S-277F (5'-GACAGTGGTCCCAAAGATGGA-3') and Als-3 (5'-GTGGATCTAGTAATGCGTTTGGG-3') to amplify a 497-bp band. RMCE events were then confirmed with five PCR analyses specific to the RMCE 5' border, RMCE 3' border, target 5' border, target 3' border, and the 5' border to 3' border full lengths of RMCE, target, and excision. The RMCE 5' border PCR used the common RMCE-specific primer Als-3 and a target line 5' border-specific primer, 53-1S1 (5'-TGTTTGTTGTTTCCAAGATTGACTGC-3'), for line A, 70-1S (5'-TCTTTCCCTCCCAGAGAGTAACAAGC-3') for line B, and 8H-ScaS1 (5'-ATAGAGGATTGGGACTCGTCGTGC-3') for line C. The RMCE 3' border PCR used the common RMCE-specific primer Cyan-1 (5'-ATGGCCCTGTCCAACAAGTTCATC-3') and a target line 3' border-specific primer, 53-1A (5'-CACCAAACTAATCATATCTCACTAAATCAATCC-3') for line A, 70-1A (5'-GCAGCGACAGGGGATTCCTCTAC-3') for line B, and 8H-VecA (5'-AGATGCTAGAAATTCAACAACGGAAGC-3') for line C. The target 5' border PCR used the same target line 5' border-specific primers and the common target-specific primer Hygro-A. The target 3' border PCR used the same target line 3' border-specific primers and the common target-specific primer Yfp-3. The full-length PCR used the same target line 5' and 3' border-specific primers to simultaneously amplify the full-length RMCE, target, and excision. The expected sizes of all PCR bands are described in the Figure 2 legend.
qPCR analyses were done on genomic DNA samples using the Taqman DNA polymerase kit with a 7500 real-time PCR system (Applied Biosystems). The relative quantification methodology and single-tube duplex PCRs, one for a target gene and the other for an endogenous control gene to normalize reactions, were used. After 2 min of incubation at 50°C to activate the Taq DNA polymerase and 10 min of incubation at 95°C to denature the DNA templates, 40 cycles of 15 s at 95°C and 1 min at 60°C were used. A soybean heat shock protein (hsp) gene was used as the endogenous control for all assays. Primers Hsp-F1 (5'-CAAACTTGACAAAGCCACAACTCT-3'), Hsp-R1 (5'-GGAGAAATTGGTGTCGTGGAA-3'), and probe Hsp-T1 (5'-VIC-CTCTCATCTCATATAAATAC-MGB-3'; Applied Biosystems) were used for the hsp control. A DNA sample known to contain one copy of the transgene to be analyzed was included as the calibrator for each qPCR assay. The scp1, hpt, ubiq10, and yfp components of QC288A were analyzed to screen for single-copy target events and to identify homozygous T1 plants. Primers/probe sets used were 35S-277F, 35S-345R (5'-CGTGGTTGGAACGTCTTCTTTT-3'), and 35S-399T (5'-FAM-CCCCACCCACGAGGAGCATCG-BHQ1-3'; Sigma-Genosis) for the scp1 assay, Hygro-591F (5'-GGATTTCGGCTCCAACAATG-3'), Hygro-659R (5'-GCCTCGCTCCAGTCAATGA-3'), and Hygro-612T (5'-FAM-CCTGACGGACAATGGCCGCATAAC-BHQ1-3') for the hpt assay, Ubq10-693F (5'-TGTGGTTGTCGACGAGTCAGTAAT-3'), Ubq10-769R (5'-GAGTTGATAAACACGACTCGTGTGT-3'), and Ubq10-719T (5'-FAM-CGGCGTCAAAGTGGTTGCAGCC-BHQ1-3') for the ubiq10 assay, and Yfp-67F (5'-AACGGCCACAAGTTCGTGAT-3'), Yfp-130R (5'-TGGTCTGCTTGCCCTTGAAG-3'), and Yfp-88T (5'-FAM-ACCGGCGAGGGCATCGGCTA-BHQ1-3') for the yfp assay. RMCE QC288A329-, target QC288A-, and donor QC329-specific qPCR assays were all designed around the FRT1 site. Primers/probe sets used were 288A-1F (5'-ATTACTATTTACAATTACAGTCGACCCAAC-3'), Als-163R (5'-GGAAGAAGAGAATCGGGTGGTT-3'), and Als-110T (5'-FAM-CCACACAACACAATGGCGGCCA-BHQ1-3') for the RMCE assay, 288A-1F, Hygro-116R (5'-TCGAAGCTGAAAGCACGAGAT-3'), and Hygro-79T (5'-FAM-CTCTCGGAGGGCGAAG-BHQ1-3') for the target assay, and 329-1F (5'-AAACGACGGCCAGTGCCAAG-3'), Als-163R, and Als-110T for the donor assay. Primers Ucp3-57F (5'-TCGAGCGGCTATAAATACGTACCT-3'), Flp-A (5'-GTCTTGCAGAGGATGTCGAACTGG-3') and probe 5'-FAM-CCTGCGCTACCATCCCTAGAGCTGC-BHQ1-3' were used for the flp QC292-specific qPCR.
We are grateful to DuPont/Pioneer colleagues Dr. Alex Lyznik for comments on the manuscript, Russ Booth for managing transgenic plants, and the soybean transformation group for producing transgenic plants. Received February 24, 2009; accepted May 5, 2009; published May 8, 2009.
The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Zhongsen Li (zhongsen.li{at}cgr.dupont.com).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.109.137612 * Corresponding author; e-mail zhongsen.li{at}cgr.dupont.com.
Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7: 649–659[CrossRef][Web of Science][Medline] Baer A, Bode J (2001) Coping with kinetic and thermodynamic barriers: RMCE, an efficient strategy for targeted integration of transgenes. Curr Opin Biotechnol 12: 473–480[CrossRef][Web of Science][Medline] Chawla R, Ariza-Nieto M, Wilson AJ, Moore SK, Srivastava V (2006) Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol J 4: 209–218[CrossRef][Web of Science][Medline] Day CD, Lee E, Kobayashi J, Holappa LD, Albert H, Ow DW (2000) Transgene integration into the same chromosome location can produce alleles that express at a predictable level, or alleles that are differentially silenced. Genes Dev 14: 2869–2880 Djukanovic V, Orczyk W, Gao H, Sun X, Garrett N, Zhen S, Gordon-Kamm W, Barton J, Lyznik LA (2006) Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems. Plant Biotechnol J 4: 345–357[CrossRef][Web of Science][Medline] Groth AC, Calos MP (2003) Phage integrases: biology and applications. J Mol Biol 335: 667–678[CrossRef][Web of Science] Horn C, Handler AM (2005) Site-specific genomic targeting in Drosophila. Proc Natl Acad Sci USA 102: 12483–12488 Iida S, Terada R (2005) Modification of endogenous natural genes by gene targeting in rice and other higher plants. Plant Mol Biol 59: 205–219[CrossRef][Web of Science][Medline] Lauth M, Spreafico F, Dethleffsen K, Meyer M (2002) Stable and efficient cassette exchange under non-selectable conditions by combined use of two site-specific recombinases. Nucleic Acids Res 30: e115 Li Z, Xing A, Moon BP, Burgoyne SA, Guida AD, Liang H, Lee C, Caster CS, Barton JE, Klein TM, et al (2007) A Cre/loxP mediated self-activating gene excision system to produce marker gene free transgenic soybean plants. Plant Mol Biol 65: 329–341[CrossRef][Web of Science][Medline] Louwerse JD, Van Lier MCM, Van der Steen DM, De Vlaam CMT, Hooykaas PJJ, Vergunst AC (2007) Stable recombinase-mediated cassette exchange in Arabidopsis using Agrobacterium tumefaciens. Plant Physiol 145: 1282–1293 McLeod M, Craft S, Broach JR (1986) Identification of the crossover site during FLP-mediated recombination in the Saccharomyces cerevisiae plasmid 2 µm circle. Mol Cell Biol 6: 3357–3367 Nanto K, Yamada-Watanabe K, Ebinuma H (2005) Agrobacterium-mediated RMCE approach for gene replacement. Plant Biotechnol J 3: 203–214[CrossRef][Web of Science][Medline] Ow DW (2002) Recombinase-directed plant transformation for the post-genome era. Plant Mol Biol 48: 183–200[CrossRef][Web of Science][Medline] Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotechnol J 2: 169–179[CrossRef][Web of Science][Medline] Srivastava V, Ow DW (2001) Biolistic mediated site-specific integration in rice. Mol Breed 8: 345–350[CrossRef] Srivastava V, Ow DW (2004) Marker-free site-specific gene integration in plants. Trends Biotechnol 22: 627–629[CrossRef][Web of Science][Medline] Tao Y, Bidney D, Gordon-Kamm WJ, Lyznik LA (2007) Modified FRT recombination sites and methods of use. U.S. Patent Application No. WO2007011733 Trinh KR, Morrison SL (2000) Site-specific and directional gene replacement mediated by Cre recombinase. J Immunol Methods 244: 185–193[CrossRef][Web of Science][Medline] Vergunst AC, Jansen LE, Hooykaas PJJ (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res 26: 2729–2734 Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J, Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) High-frequency homologous recombination in plants mediated by zinc-finger nucleases. Plant J 44: 693–705[CrossRef][Web of Science][Medline]
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