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Plant Physiol. (1994) 106: 447-458 Seed-Specific Gene Activation Mediated by the Cre//ox Site-Specif ic Recombination System Joan T. Odell*, Joyce1. Hoopes, and Wilfred Vermerris' Agricultural Products, DuPont, Experimental Station, P.O. Box 80402, Wilmington, Delaware 19880-0402 ~~ ~ ~ recognized and recombined by Cre. Test genes have been designed such that Cre-mediated excision or inversion of loxbounded DNA sequences lead to their activation or inactivation. A lox-bounded coding region placed in inverted orientation with respect to the regulating promoter was expressed after inversion (Dale and Ow, 19.90). A loxbounded fragment placed between a promoter and coding region, disrupting gene expression, was excised to restore activity (Odell et al., 1990; Bayley et al., 1992). Translocation between lox sites located on separate chromosomes also brought together a promoter and coding region, resulting in gene activation (Qin et al., 1994). Excision of an entire gene located between lox sites resulted in loss of expression (Dale and Ow, 1991; Russell et al., 1992). These alterations in the function of a test transgene occurred after the expression of a cre gene, which was introduced either by retransformation of tissue containing the lox construction or by genetic crossing of lox- and cre-containing plants. In all previous examples Cre was expressed from a relatively constitutive promoter, the 35s promoter of CaMV. Alteration in test gene expression was assayed in roots, stems, leaves, and/or germinating seedlings. Cre-mediated gene activation in a specified tissue may provide a useful tool for developmental analyses. For example, Cre may be used to specifically activate a cell-lethal gene to determine the effects of the loss of the affected cells on surrounding tissues. Similar tissue interactions may be studied by controlling the activation of a gene whose product restores function in a mutant host. Here we examine tissuespecific regulation of gene expression mediated by Crellox. A silent GUS gene is activated by Cre-mediated excision of a lox-bounded fragment located between the promoter and coding region. The seed-specific promoters from the CY' subunit of soybean (Glycine max) P-conglycinin and from French bean (Phaseolus vulgaris) P-phaseolin were used to regulate GUS and Cre expression. These developmentally controlled systems allow an analysis of the timing and efficiency of Cremediated gene activation. Comparisons are made to pattems of activation induced by Cre expressed from the 35s promoter and by Cre protein that is targeted to the nucleus. l h e Cre//ox site-specific recombination system was used to activate a transgene in a tissue-specific manner. Cre-mediated activation of a 8-glucuronidase marker gene, by removal of a loxbounded blocking fragment, allowed the visualization of the activation process. By using seed-specific promoters, the timing and efficiency of gene activation could be followed within the developing tobacco (Nicotiana tabacum) embryo. To serve as a basis for analyzing gene expression after Cre-mediated activation, the timing and patterns of expression of the promoters of the genes encoding French bean (Phaseolus vulgaris) &phaseolin and the a' subunit of soybean (G/ycine max) &conglycinin, as well as the cauliflower mosaic virus 35s promoter, were studied in developing transgenic tobacco embryos using the same visual marker. These seed-specific promoters were expressed earlier than anticipated. l h e 35s promoter was expressed earlier than the seed-specific promoters, but not in globular-stage embryos. Cre-mediated gene activation occurred approximately 1 d after promoter activation, based on developmentalstaging, and spread progressivelythroughout the embryo. l h e timing of gene activation was varied by altering Cre expression. Efficient Cre expression ultimately directed gene activation throughout the model tissue, whereas inefficient Cre expression resulted in mosaic tissue. limited gene activation provides a system for cell lineage and developmental analyses. Site-specific recombination systems derived from yeast plasmids and bacteriophage have been shown to function in plant cells. In each case the recombinase locates and interacts with its target sites, resulting in excision of DNA sequences located between directly oriented target sites or inversion of sequences located between inverted target sites. Integration between target sites on separate DNA molecules can also occur. The site-specific recombination systems expressed in plants thus far include FLP/FRT (recombinase/recognition site) of the Saccharomyces cerevisiae 2p plasmid (Lyznik et al., 1993; Lloyd and Davis, 1994), R/RS of the Zygosaccharomyces rouxii R 1 plasmid (Onouchi et al., 1991), Gin/& of bacteriophage Mu (Maeser and Kahmann, 1991), and Crellox of bacteriophage P1 (Dale and Ow, 1990; Odell et al., 1990); all systems are reviewed by Odell and Russell (1994). The two elements of the Crellox system, Cre Kauses recombination) protein and loxP @cus of crossing over, 5, P1; also lox) sites, function well as stable components of the plant genome. loxP sites integrated in the plant genome are Abbreviations: Cab, Chl alb-binding protein; CaMV, cauliflower mosaic virus; dap, days after pollination;GUS, @-glucuronidase;Nos, nopaline synthase; npt I and npt 11, neomycin phosphotransferase I and 11; Ocs, octopine synthase; ppb, parts per billion; SV40, simian virus 40. ' Present address: Department of Genetics, North Carolina State University, Raleigh, NC 27695-7614. * Corresponding author; fax 1-302-695-4296. 447 Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 448 Odell et ai. MATERIALS A N D METHODS Chimeric Cene Constructions The French bean (Phaseolus uulgaris) @-phaseolinpromoter (Ph/P) and the soybean (Glycine max) a’ subunit of 8conglycinin promoter (Cg/P; Doyle et al., 1986) were provided by J. Slightom. NcoI sites had been added at each translation initiation site while preparing the promoter fragments using the PCR technique (described by Slightom and Chee, 1991). Plasmids containing the promoter fragments also contained a 1.17-kb fragment including the phaseolin 3’ polyadenylation signal region (Ph 3’), downstream of the NcoI site. A linker was added to create the following sites between the promoters and 3‘ regions: NcoI, SmaI, Asp718, and XbaI. The cre coding region was then added as an NcoIXbaI fragment to prepare the chimeric genes, Ph/P-cre and Cg/P-cre (Fig. 1A). Each gene was transferred as a HindIII fragment into the binary vector pZS94. This plasmid contains A +Ph/P( + + C 3 cre CR/PI c r e 5 4 Ph/PI I~ c r~e Gus IPh 3+IPh Y+- IOCS 3’+ IPh 3’1- --I 3 5 s c I Figure 1. Constructions of chimeric cre and CUS genes. A, Cenes to express the cre coding region from t h e P-phaseolin promoter (Ph/P), a‘ @-conglycininpromoter (Cg/P), or 35s promoter with the Cab leader (35SC). T h e NTcre coding region includes t h e SV40 Tantigen nuclear localization signal. T h e 3’ end is either from the 8phaseolin gene (Ph 3’) or the Ocs gene (Ocs 3’). B, lnterrupted /ox2-CUSgenes designed to be inactive. /ox sites (filled arrowheads) surround a fragment including a polyadenylation signal sequence (polyA).C, Control genes that express CUS from the promoters in A. One has the 3’ end from the Nos gene (Nos 3’). D, Cre-mediated activation of t h e /ox2-CUSgene. Recombination between directly repeated lox sites results in excision of the segment between the two sites, leaving one /ox site in the untranslated leader of the CUS mRNA. Plant Physioi. Val. 106, 1994 the origin of replication and ampicillin resistance gene from pBR322!, the replication and stability regions of t he Pseudomonas ae~~uginosa plasmid pVSl (Itoh et al., 1984), and T-DNA borders described by van den Elzen et al. (1985). To provide a plant transformation selection marker, a PstI-llsp718 fragment containing a mutant 35s-acetolactate sy nthase gene that confers sulfonylurea resistance (described by Russell et al., 1992) was added. A gene encoding Cre with a nuclear localization signal added at the N terminus was prepared. At the NcoI site in Cg/P-cre an oligonucleotide encoding amino acids 118 through 123 and 126 through 132 of the SV40 laige T antigen (Rihs and Peters, 1989; Fig. 1A) was inserted. ‘rhe oligonucleotide was designed so that the 3’ NcoI site was not regenerated. The resulting Cg/P-NTcre gene was transferred as a HindIII fragment into pZS96. pZS96 is the vector pZS94 with a Nos/P-nptII-Ocs 3’ gene added to serve as a kanamycinresistant plant selection marker. A gene containing the 35s promoter of CaMV and the cre coding region was constructed (Fig. 1A).A promclter fragment containing the 355 promoter and leader from the Chl a/bbinding protein gene 22L (35SC; Harpster et al., 1988) was joined to the cre coding region at the NcoI site located at the translaiion-initiation ATG. The cre coding regon was followed by a 720-bp fragment containing the polyadenylation signal sequence from the Ocs gene (Ocs 3’). An EcoRI-Asp718 fragment containing the entire 35SC-cre gene was transferred to the binary vector pZS198. pZS198 is pZS96 with the bacterial ampicillin-resistanceselection marker replaced by a bacterial kanamycin-resistance selection marker (nptI; kindly provided by M. Locke). Inactive marker GUS genes with a lox-bounded blocking fragment (Iox’) between the promoter and coding; region were constructed as follows (Fig. 1B). Before adding ihe Iox2 fragment to each promoter, the NcoI site at the 3’ end of each promoter was deleted so that there would be no translationinitiatirig ATG 5’ to the first lox site. Plasmids containing each promoter were digested with NcoI, treated with S1 nuclease to remove the overhanging ends, and then religated (Ph/P) or cut at the SmaI site downstream of the NcoI site and religated (Cg/P). The 3’ end of each promcter fragment was sequenced to verify that the ATG from the VcoI site was removed. A linker containing XhoI and NcoI sitw was added between the Asp718 and XbaI sites located 3’ to the Cg/P. The lox-polyA-lox fragment described by Odell et al. (1990) was added as a XhoI-Ncol fragment, after an NcoI linker was added at the HindIII site at the 3’ end. This fragnient contains the polyadenylation signal sequence region frcm a tobacco Rubisco small subunit gene (Mazur and Chui, 15185) between directly oriented lox sites. Both lox sites are orirtnted so that the two ATGs present in the site are in reverse orientation with respect to the promoter. The GUS coding r2gion (Jefferson et d.,1987) was joined, via an NcoI site at its translation start site, 3’ to lox’. An Asp718-Asp718 fragment from this clone containing lox-polyA-lox-GUSwas added io the Asp718 site between the Ph/P and Ph 3’, and clones with the desired fragment orientation were identified. The entire Ph/P or Cg/ P-lox-poIyA-lox-GUS-Ph3 ’ gene was transferred as a HindIII fragment into the binary vector pZS96. Control Ph/P-GUS and Cg/P-GUS genes wlzre also con- Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Crellox-Mediated Seed-Specific Gene Activation structed (Fig. 1C). An NcoI-Asp718 fragment containing the GUS coding region was added between the NcoI and Asp718 sites between the Cg/P or Ph/P and Ph 3' in plasmids described above for the cre constructions. A HindIII fragment containing each gene was transferred into the pZS96 vector. Plant Transformation All binary vectors containing cre or lox constructions were transferred into Agrobacterium tumefaciens strain LBA4404 by three-way matings (Ditta et al., 1980), direct DNA uptake (An et al., 1988), or electroporation (Mersereau et al., 1990). Selected transformed A. tumefaciens were used to obtain Nicotiana tabacum cv Xanthi transformants as described by Odell et al. (1990) using either 300 pg/mL kanamycin or 50 ppb chlorsulfuron for selection, depending on the selection marker present. Seed Germination Assay Seed was sterilized and assayed for seedling resistance as described previously (Odell et al., 1990) using 200 mg/L kanamycin or 50 ppb chlorsulfuron. 449 Southern Blot Analysis Preparation of DNA was based on the procedure of Shure et al. (1983), with modifications by J. Chen. Fresh tissue was ground in a mortar with liquid nitrogen. Buffer containing 7 M urea, 0.31 M NaCl, 50 IT~MTris, pH 8.0, 20 mM EDTA, and 1% sarkosyl was added and the sample was shaken vigorously then incubated at room temperature for 10 min. The sample was then extracted with an equal volume of pheno1:chloroform:isoamylalcohol at 25:24:1. The supematant was passed through Miracloth, then 0.1 volume of 4.4 M NH4Ac and 0.5 volume of isopropanol were added. The DNA was spooled, washed with 80% ethanol, dried, dissolved in water, and precipitated a final time. Approximately 10 pg of DNA was digested with Asp718 or HindIII, and the resulting fragments were separated by gel electrophoresis then transferred to a Hybond-N (Amersham) filter. Hybridization was to a digoxigenin-labeled probe, which was detected by chemiluminescence using Lumi-Phos 530 (Genius System, Boehringer Mannheim). RESULTS Experimental Design for Seed-Specific Gene Activation Cross-pollinations Hand pollinations were performed as described previously (Odell et al., 1990). Initially, single-locus homozygous cre plants, identified through seed-germination assays, were crossed with primary /ox2-GUStransformants that contained one or more (two or three) kanamycin-resistance loci. In this case, all seed inherit the cre gene, but 50% or less do not inherit the lox2-GUSgene. Therefore, some F1 embryos would never show GUS activation, having not inherited the GUS gene. In cases in which GUS was expressed in some F1 embryos, there were also embryos from the same cross that did not show GUS activity as expected; these were not included in figures. In cases in which no GUS activity was detected, such as in very early-stage embryos, the embryo sample size was large enough to assure that embryos inheriting /ox2-GUSwould have been included in the assay. Single-locus homozygous /ox2-GUS transformants were then identified through seed-germination assays. These were crossed with primary transformants bearing 35SC-cre or Cg/ P-NTcre genes. The same inheritance situation described above occurs in the F1 seed but is reversed for the cre and lox*-GUS genes. Seed Dissection and Analysis Pods were harvested at various days after selfing (in the case of plants containing Ph/P-GUS, Cg/P-GUS, and 35S/ P-GUS transgenes) or cross-pollination (dap). Seed was removed from the pod, dissected to separate the embryo from the endosperm (which often remained partially attached to the seed coat), placed directly into GUS enzyme assay buffer (Jefferson, 1987), and incubated at 37OC. Samples were examined for GUS staining at various times, and photomicrographs were taken using a Zeiss Stemi SR dissecting microscope, or a Wild M8 dissecting microscope for the higher magnifications. To assess the timing and efficiency of Cre-mediated recombination in plant cells, we used the Crellox system to activate a marker GUS (Jefferson et al., 1987) gene in developing seed. An inactive GUS gene was constructed by placing a /ox-bounded DNA fragment (represented by /ox2), containing a polyadenylation signal sequence, between the promoter and coding region (Fig. 1B). The directly repeated lox sites provide the substrate for Cre-mediated excision of the blocking fragment, leading to expression of GUS (Fig. 1D). Cremediated recombination of the lox2-GUS gene in vitro did give the expected products. This activatable marker and cre were regulated by two seed-specific promoters, derived from genes encoding French bean ,&phaseolin (referred to as 0phaseolin promoter and Ph/P) and the soybean a' subunit of P-conglycinin (referred to as a' /3-conglycinin promoter and Cg/P). When this work was initiated, the patterns of expression of these two promoters in regions of the embryo and in the endosperm of transgenic tobacco seed were not well established, and we expected different patterns for their expression. Control GUS genes were constructed without the intervening lox-bounded fragment (Fig. 1C). For comparison, cre was also expressed from the CaMV 35s promoter. The timing of marker gene expression after activation and the extent to which it is activated throughout the embryo were determined. These analyses characterize the Crellox recombination process in plant cells. Preparation of Transgenic Plants Each lox-inactive chimeric GUS gene and control GUS gene was stably integrated into the tobacco genome through A. tumefaciens-mediatedtransformation. A linked nptII gene, conferring kanamycin resistance, was used to select the transformants and to identify lines carrying a single integrated locus. Lines were screened for GUS activity in mature seed. Of 12 kanamycin-resistant transformants with Ph/P-GUS or Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 450 Odell et al. Cg/P-GUS in the introduced T-DNA, only 2 lacked GUS activity in mature seed. In these 2 lines an intact GUS gene may not have been integrated. Of 15 kanamycin-resistant lines transformed with a GUS gene interrupted by the /oxbounded fragment, 8 showed no GUS activity, whereas 7 expressed a very low level of GUS activity in mature seed relative to the Ph/P-GUS and Cg/P-GUS levels. Overnight incubation in GUS assay buffer was required to detect light blue staining, whereas staining began to appear within 15 min in seed with no blocking fragment. In the 8 negative lines the intervening /ox-bounded fragment appeared to be effective in blocking expression of the GUS coding region, although it is possible that a few of them did not integrate an intact GUS gene. Seven of these lines were tested for Cre activation of GUS as described below. In all but 1, GUS expression could be activated, verifying the presence of the intact gene. The blocking fragment was not completely effective, as evidenced by the 7 lines with low GUS activity. These were not examined to determine whether the "inactive" GUS gene construction was transferred in an intact form. Separate tobacco lines carrying chimeric seed promoter-cre genes were selected using a sulfonylurea-resistant acetolactate synthase marker. By seed-germination assays on chlorsulfuron, four single-locus lines were identified, then homozygotes in these lines were identified. Of these four lines, three were able to activate the inactive GUS gene as described below. In the fourth line an active ere transgene may not have been integrated. In the course of these experiments, two different gene constructions containing the 35S promoter and ere coding region were used. Homozygous tobacco plants with a 35Scre transgene were produced and assayed in previous studies (cre/Hpt-A, Odell et al., 1990; Russell et al., 1992). This chimeric gene has an untranslated leader composed of sequence from the leader of the 35S RNA of CaMV (21 bp), and sequence upstream of the translation start site of the bacteriophage PI ere gene (about 50 bp). In a second gene, called 35SC-cre, the leader of the petunia Cab gene follows the 35S promoter, and is joined to the start of the ere coding region by an Ncol site (see "Materials and Methods"). Tobacco transformants containing the 35SC-cre transgene were selected using a linked kanamycin-resistance selection marker. Other control plants bearing a GUS gene expressed from the same 35S promoter linked to the Cab leader (35SC-GUS; Fig. 1C) were produced and described previously (Russell et al., 1992). Plant Physiol. Vol. 106, 1994 opment, although plants were grown under the same conditions. For example, the heart to small torpedo-stage embryos in Figure 2C were collected at 9 dap and those of similar stages in Figure 2A were collected at 10 dap. More advanced torpedo-stage embryos were also on occasion found at 10 dap, such as those in Figure 3F. There was also substantial variation in development among seed within the same pod. For example, all of the embryos in Figure 3A, which range from torpedo to maturing cotyledon stages, were dissected from seed within the same pod collected at 14 dap. Thus, when assaying individual embryos it is more informative to *•** Figure 2. Expression of GUS activity from /3-phaseolin, a' /3-conglycinin, and 35S promoters in tobacco embryos and endosperm. All embryos are shown at the same magnification except for those in Expression of Ph/P, Cg/P, and 35SC/P All Initiate at the F, bottom row, and G, as indicated below. All samples were incuHeart Stage bated in GUS assay buffer overnight at 37°C unless otherwise specified. A, Embryos containing the Ph/P-GUS gene dissected 10 Previously, expression of the 0-phaseolin, a' /3-conglycidap. B, Endosperm of seed containing Cg/P-GUS dissected 10 dap. nin, and 35S promoters in developing transgenic tobacco C, Embryos containing Cg/P-GUS dissected 9 dap. D, Embryos seed had been determined only by bulk seed analyses. Since containing Cg/P-GUS dissected 11 dap and incubated for 2 h in we planned to visualize GUS staining of individual embryos GUS assay buffer. E, The same embryos as in D incubated overnight. to analyze Cre-mediated gene activation, it was critical to F, Embryos containing 35SC-GUS dissected at 10 dap. The upper determine the expression patterns of these promoters using set is shown at the same magnification as those in A-E. The lower the same procedure. Seed bearing Ph/P-GUS, Cg/P-GUS, or set contains the same embryos shown at 1.6x higher magnification 35SC-GUS were dissected at different dap and the embryos for better visualization. G, Embryos containing 35SC-GUS dissected and endosperm were stained separately for GUS enzyme at 10 dap and incubated in GUS assay buffer for 2 h, shown at 1.6x activity. Variation occurred in the timing of embryo develhigher magnification than those in A to E. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Cre//ox-Mediated Seed-Specific Gene Activation 451 B A Figure 3. GUS activity following Cre-mediated activation of /ox2-GUS genes. All samples are from Fi seed unless otherwise specified. A, Embryos inheriting Cg/P-/ox2-GUS and Cg/P-cre dissected 14 dap. Inset, Embryos inheriting Ph/ P-/ox2-GUS and Ph/P-cre dissected 12 dap. B, Endosperm of seed inheriting Ph/P-/ox2-GUS and Ph/P-cre dissected 11 dap. C, Embryos inheriting Ph/P-/ox2-GUS and 35S-cre dissected at 13 dap. Inset, Embryos inheriting Ph/P-/ox2-GUS and 35S-cre dissected at 12 dap. Note that these embryos are inverted. D, Endosperm of seed inheriting Ph/P-/ox2-GUS and 35S-cre dissected at 16 dap. E, Embryos inheriting Cg/P-/ox2-GUS and 35S-cre dissected at 12 dap. F, F2 embryos from FI plants grown from F, seed that inherited Ph/P-/ox2-GUS and Ph/P-cre dissected 10 dap. describe stages of embryo development than dap. In Figures Figure 2, F and G, and Figure 4 are shown at higher magni2 and 3, embryos are shown at the same magnification (except fication for better visualization. in Fig. 2, F and G) so that the stages of development can be The expression patterns of the /3-phaseolin and a' ficompared by the size and shape of the embryos. Those in conglycinin promoters were similar throughout seed develFigure 2A are entering the heart stage or are hearts or young opment. No GUS activity was observed in globular-stage torpedos. Figure 3A includes embryos of torpedo and early embryos. As embryos entered the heart stage, spots of GUS to maturing cotyledon stages. Embryos in the bottom row of activity were observed just below the cotyledonary lobes (see Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 452 Plant Physiol. Vol. 106, 1994 Odell et al. Cg/P ioxp H——W. H .5 -s A Ph 3' loxP 1.6 - 5.0 kb -3.3 kb X + Cre Cg/P ioxp + H •=> A GUS Ph 3' - 3.5 kb -1.8 kb 1 2 3 4 Figure 4. Molecular analysis of Cre-mediated recombination leading to activation of CUS gene expression. A, Diagram of the inactive Cg/P-/ox2-GUS gene (above) and Cre-activated Cg/P-CUS gene (below). Shown below each diagram are the DNA fragments produced by H/ndlll (H) or Asp718 (A) digestion from the original construction, and those predicted following recombination. The hatched bar indicates the CUS coding region probe used to detect these fragments. Sizes shown are approximate. Abbreviations are as described in Figure 1. B, Southern blot showing bands from the inactive and recombined genes. Lanes 1 and 3, Wild-type tobacco DNA spiked with inactive Cg/P-/ox2-CUS gene-containing plasmid. Lanes 2 and 4, DNA from an f, ere x /ox plant. DNA samples in lanes 1 and 2 were digested with Asp718, and those in lanes 3 and 4 were digested with H/ndlll. Left and right upper arrows mark the bands representing nonrecombined fragments in lanes 1 and 3, respectively. Left and right lower arrows mark the expected recombination products in lanes 2 and 4, respectively. The upper band in lane 4 is likely to result from a rearrangement of integrated DNA. The positions of size markers are shown to the left. Fig. 2A). More spots accumulated in this area, creating a ring stage embryos that stain entirely blue during longer incubaof GUS activity in the transition zone between the developing tions (Fig. 2, D and E). Thus, this region, which is the first to show phaseolin and conglycinin promoter expression at the cotyledons and embryo axis of heart-stage embryos. This ring then spread up into the cotyledons and down into the axis, heart stage, maintains the highest level of expression more rapidly on the outside edges (Fig. 2, A and C). Embryos throughout later stages of development. entering the torpedo stage lacked GUS activity only in the The 35SC promoter showed a different pattern of exprestips of the cotyledons and the bottom of the root axis (Fig. 2, sion, observed in the three independent transformants bearA and C). Torpedo-stage embryos stained entirely blue. With ing the 35SC-GUS gene that were analyzed. Surprisingly, GUS activity was also absent in globular embryos. These both promoters, GUS activity was absent in the endosperm of very early-stage embryos but then spread throughout this unstained globular embryos are not shown due to the diffitissue (Fig. 2B). The endosperm expression was unexpected culty of photographing them under the conditions we emfor the a' /3-conglycinin promoter, since no activity was seen ployed. GUS activity appeared and spread more rapidly than in soybean endosperm using in situ analysis (Perez-Grau and when expressed by the phaseolin and conglycinin promoters. Goldberg, 1989). Embryos entering the heart stage showed a patch of GUS The embryo expression of both promoters was much earlier activity in the top central region (Fig. 2F). The expression than is generally described for seed storage proteins. Therespread rapidly; mature heart-stage embryos stained entirely fore, eight independent transformants containing Ph/P-GUS blue. This timing varied slightly between independent transand four containing Cg/P-GUS were assayed. All but one formants, but was always substantially earlier than observed showed strong endosperm expression and similar patterns of for the phaseolin and conglycinin promoters. GUS activity embryo expression. In several lines the timing varied slightly, also developed in the endosperm. After a short incubation in being either a bit more rapid or delayed than that described GUS assay buffer, the tips of the cotyledons and the lower above. One exceptional Ph/P-GUS line showed much repart of the axis remained unstained, indicating that these are duced expression; spots of GUS activity appeared first in the regions with the weakest promoter expression (Fig. 2G). torpedo stages, which stained entirely blue in all of the other The short incubation also allows the detection of a spot of lines (data not shown). Mature embryos of this line still did strong GUS activity at the tip of the root axis, a pattern not not stain solidly blue, but were blotchy, indicating reduced observed in the /3-phaseolin and a' /3-conglycinin promoters. GUS expression. This pattern could be explained by greatly reduced functioning of the phaseolin promoter, due to its Cre-Mediated Gene Activation position of integration in the tobacco genome in this particOnce we determined the timing and pattern of GUS expresular line. sion directed by the /3-phaseolin and «' /3-conglycinin proIncubation of embryos in GUS assay buffer for a short moters in individual embryos, we could compare it to the period of time allows the localization of regions with strongest GUS expression seen after Cre-mediated activation of the GUS expression. The cotyledon-axis junction region is always /o*2-GUS gene. Cre and lox elements were joined in FI seed first to stain for GUS activity,Downloaded even in torpedoand laterfrom on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Cre//ox-Mediated Seed-Specific Gene Activation by crossing a plant containing a ere gene and a plant containing a /OA:2-GUS gene. Seeds of varying developmental stages were dissected and the embryos and endosperm were stained for GUS enzyme activity. GUS activity was observed in embryos and endosperm of seed derived from crosses between lox and ere plants, indicating that the /<w2-GUS gene was activated, whereas seed from crosses between lox plants and control wild-type plants never showed any GUS staining. Activation of the /o;t2-GUS gene by Cre-mediated recombination, diagrammed in Figure ID, was later verified in Ft plants. DNA of ere X lox F] plants analyzed on Southern blots showed GUS probe-hybridizing bands of the sizes that would be expected after excision of the DNA segment between the lox sites (Fig. 4). Similar timing and patterns of GUS activation were observed in seed derived from crossing four independent Ph/ P-/ox2-GUS transformants with a Ph/P-cre plant, and two independent Cg/P-/ox2-GUS transformants with two independent Cg/P-cre transformants (all four combinations). In each case no GUS activity was observed in heart-stage or young torpedo-stage embryos. As described above, we unexpectedly found that the /3-phaseolin and a' /3-conglycinin promoters are active during these stages. It was critical to visualize this early expression of these promoters to determine that a delay in expression occurred when recombination between lox sites was required to activate the GUS gene. The first few spots of GUS activity appeared in torpedo-stage embryos (Fig. 3A). This represents a delay of about 1 d in the developmental time course. Spots of GUS activity occurred first at the cotyledon-axis junction, which is the region of earliest and highest /3-phaseoIin and a' /3-conglycinin promoter expression (see Fig. 2, A-D). The GUS-expressing region then spread up into the cotyledons and down into the axis, until the whole maturing cotyledon-stage embryo stained blue. Activation occurred coordinately in the endosperm, initially in spots of activity that then spread throughout the endosperm (Fig. 3B). No difference in the timing of GUS gene activation was observed in FI embryos derived from reciprocal crosses (data not shown). 453 magnification). The 35S-cre plants were from lines analyzed in previous experiments in which the transformation selection marker, bounded by lox sites, was excised from the tobacco genome in ere by lox F, progeny (Russell et al., 1992). Line Cre3 was the best at directing recombination in these previous experiments, and line Cre2 was the least effective. Crosses of /ox2-GUS plants (both Ph/P-/ox 2 -GUS and Cg/P-/ox2-GUS plants used) with two plants from line Cre3 produced an activation pattern in F] seed similar to that described above for Ph/P-cre and Cg/P-cre activation (Fig. 3A): a delay of about 1 d following initial expression of phaseolin and conglycinin promoters (Fig. 3E, and earlier stages shown in Fig. 5B at higher magnification). This result indicates that the GUS gene was not preactivated. In crosses of /ox2-GUS plants with plants from line Cre2, FI seed showed a pattern of GUS expression that indicated even later excision of the /oxbounded blocking fragment. No GUS activity was observed until embryos reached a late torpedo stage. Spots of GUSexpressing cells were observed first in the cotyledon-axis B Eliminating the Delay in Gene Activation When the same developmentally regulated promoters direct expression of Cre and GUS, the time that is required for Cre expression and the recombination process itself is seen as a delay in GUS expression. Earlier expression of Cre could eliminate this delay. We initially thought that the 35S promoter would be expressed throughout embryo development in a more constant manner than are the seed-specific promoters. If this were true, Cre protein would be present and able to create the activated GUS gene prior to the time when the seed-specific promoters are expressed. Therefore, GUS activity would appear with the same timing as the expression of Ph/P-GUS. Surprisingly, this was not the case in initial experiments. Figure 5. Comparison of CUS gene activation by Cre expressed When plants containing Ph/P-/ox2-GUS or Cg/P-/ox2-GUS from different ere genes. These F, embryos are shown at 1.6X the were crossed with several different plants containing the magnification of those in Figures 2 and 3. A, Embryos inheriting Cg/ 35S-cre gene (not 35SC-cre, see "Preparation of Transgenic P-/ox2-GUS and 35SC-cre dissected at 10 dap. B, Embryos inheriting Plants'), two different patterns of GUS expression were obCg/P-/ox 2 -GUS and 35S-cre dissected at 10 dap. C, Embryos inherserved in the F! seed (Fig. 3, C and E; embryos at same iting Cg/P-/ox2-GUS and Cg/P-NTcre dissected at 11 dap. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Odell et al. 454 junction region, and then in the cotyledons. In older embryos there were large patches as well as smaller spots of GUSexpressing cells. Endosperm from maturing seed also had localized spots of GUS activity. Surprisingly, one plant from the Cre3 line caused this same delayed and limited pattem of GUS activation (Fig. 3, C and D). This plant appeared to have reduced Cre expression. Its Cre3 siblings activated GUS expression no earlier than when Cre was regulated by the conglycinin and phaseolin promoters. The gene construction that includes the 35s promoter/Cab leader and cre coding region, 35SC-cre, did direct the earlier activation of the GUS gene that was originally predicted. In Fl embryos from crosses of one Cg/P-lox2-GUS plant with two independent 35SC-cre transformants, GUS was expressed in embryos entering the torpedo stage in all but the tips of the cotyledons and bottom of the axis. Shown in Figure 5A, these embryos are at higher magnification, but are of similar stages as some in Figure 2, A and C. The tip of the root axis did show GUS activity, following the expression pattern specific for the 355 promoter. Young torpedo-stage embryos stained entirely blue. This mimics the expression pattem for Cg/P-GUS (no activation required), with the addition of the blue axis tip. Thus, the blocked GUS gene appears to have been preactivated by Cre present in heartstage embryos, as predicted from the expression pattem of this same promoter visualized using the GUS marker, 35SCGUS, described above. From the patterns of GUS gene activation, we infer that the initial 35s-cre construction directed later and lower levels of expression than 35SC-cre, which probably expressed in a manner similar to 35SC-GUS. The better expression of the 35SC-cre gene, as compared to the 35s-cre gene, may be attributed to the different 5’ untranslated leader sequences. In particular, the sequence surrounding the translation initiation ATG of the 35SC-cre gene is more similar to the consensus sequence desired for maximal translatability (Fig. 6; Kozak, 1986). It is not known if translational efficiency accounts for the difference in expression levels, or whether increased mRNA stability is possibly conferred by the Cab leader as well as other unknown factors. Nuclear Targeting of Cre The efficiency of Cre-mediated recombination could potentially be increased by targeting Cre protein to the nucleus. TTAAATGTCC c r e L-c re I I AACAAT G CT con sensus I l l I I AACCATGGCC CabL-cre Figure 6. Comparison of sequences surrounding t h e translation initiation ATG in two different cre genes expressed from t h e 35s promoter. The top sequence is from t h e 35s-cre gene, which has the natural sequence 5’ to cre in the P1 genome upstream of the ATC (creL-cre).The bottom sequence is from the 35SC-cre gene, which has the leader from the Cab gene 22L (CabL-cre).The middle sequence is the Kozak consensus for maximal translation, with the most important nucleotides underlined. Vertical dashes indicate the identities between t h e sequences surrounding t h e ATGs. Plant Physiol. Vol. 106, 1994 Cre is a small (38 kD) prokaryotic protein that obviously is able to enter the eukaryotic nucleus. However, by facilitating entry into the nucleus, the delay in timing between Cre expression and lox gene activation might be decreased. This hypothesis was tested by placing the SV40 large T-antigen nuclear-localizationsignal sequence at the N temunus of Cre expressed from an NTcre gene (see “Materialsand Methods”). Three independent Cg/P-NTcre transformants were crossed to a Cg,lP-lox2-GUSline, and GUS expression was followed in F1 einbryos to determine whether activation was more rapid than in embryos inheriting Cg/P-cre. The same I-d delay in GUS expression was observed with boih cre genes (Fig. 3A, and Fig. 5C at higher magnification). Thus, the additiori of this targeting signal did not increase the in vivo efficiency of the Crellox recombination system. Loxz-CIJS I s Preactivated in Fz Seed To determine whether the blocked GUS gene w as activated in genetically effective cells in the F1 generation, plants were grown from Fl seed derived from crosses between Ph/P-lox2GUS plants and Ph/P-cre or 35s-cre plants. GUS expression was observed in developing F2 seed produced by selfing each F1 plant. Preactivation of the GUS gene in the Fl generation, prior to F2 seed formation, can be determined in two ways. The GUS expression pattern in F2 embryos should not be delayed, as it is in the F1 embryos, if preactivation occurred. Also, genetic segregation predicts different ratios of GUSexpressing embryos for preactivation versus actixration in the FZseed. Each of the original cre parents crossed to produce F1 seed was homozygous for the cre gene at a single locus. Each of the orignal lox parents was hemizygous (primary transformants) at a single locus for the interrupted GUS (lox2-GUS) gene. Therefore, each cross produces 50% of seeds inheriting both crie and lox, and 50% of seeds inheriting only cre. Seedlings that inherited both cre and lox were selected by their resistance to kanamycin, the marker linked to the lox2GUS gene. Each of these F1 plants is heterozygous for both cre and lox. Selfing of the F1 plant produces F2 seed in the following ratios of genotypes: 9 lox cre:3 lox alone:3 cre a1one:l neither lox nor cre. If preactivation occurred in the F1 generation, cre is not necessary for activation in F2 seed, so all Fz seed inheriting lox would express GUS. A ratio of 3 b1ue:l white embryos is expected in this situation. If activation is necessary for GUS expression, then cre must be inherited along with lox2-GUS,so a ratio of 9 blue:7 wbite embryos is expected. Ratios of 3 b1ue:l white were obsemed in the F2 embryos of each selfed cre-lox F1 plant in samples of over 40 (for example, 31 blue embryos:12 white embryos). Thus, the genetic analysis indicates that the lox blocking fragment is removed in genetically effective cells prior to F2 seed development. Results of the second assay for preactivation were consistent with the genetic data in that the timing of GU!; expression in F2 embryos was not delayed. Similar express’on patterns were observed in F2 embryos of two individml F1 plants derived from each of the following crosses: two independent Ph/P-lox2-GUS lines with one Ph/P-cre line and one Ph/Plox2-GUSline with two 35s-cre lines. Spots of GUS activity Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. + Crellox-Mediated Seed-Specific Gene Activation appeared first in heart-stage embryos (not shown) and a thick blue ring was seen in mature hearts (Fig. 3F), clearly earlier expression than seen in the F, embryos. The expression level appeared to be slightly reduced, since in young torpedo-stage embryos the axis stained a lighter blue and the axis tip remained white, even after the overnight incubation in GUS assay buffer. Reasons for this reduced expression are unknown. DISCUSSION To characterize the timing of Cre-mediated gene activation in seed, it was first necessary to establish the expression patterns of the two seed-specific promoters used in our system. By staining intact embryos we were able to visualize the timing and pattem of expression of the marker GUS gene directed by the promoter of either the French bean P-phaseolin gene or the soybean a‘ subunit of the P-conglycinin gene. In each case the expression was earlier in the development of transgenic tobacco embryos than expected from previous reports. Soybean P-conglycinin genes (a’, a, and P subunits) are highly expressed in soybean embryos in midto late-maturation stages (Meinke et al., 1981; Walling et al., 1986). In situ hybridization analysis of P-conglycinin expression (both a’ and /3 subunit mRNAs hybridized to the probe) showed no expression in globular-, heart-, or cotyledon-stage soybean embryos (Perez-Grau and Goldberg, 1989). Mid- to late-maturation-stage embryos expressed ,8-conglycinin in both cotyledons and the axis. In this analysis, there also was no expression in the endosperm. Although seed development in tobacco and petunia is much more rapid than in soybean, previous results indicated that transgenic tobacco and petunia plants expressed the a’ P-conglycinin promoter in the corresponding mid to late stages of embryo development (Beachy et al., 1985; Chen et al., 1988; Naito et al., 1988; Chamberland et al., 1992). Since promoter sequences to -257 bp upstream of the transcription start site were shown to be sufficient for temporal control, as well as for maximal expression (Chen et al., 1986, 1988), our 550-bp promoter fragment was sufficient to contain all of the regulatory sequences. Using GUS staining of individual embryos, we observed expression in heart-stage embryos. Since the same heart-stage expression was observed in embryos of many independent transformants, this pattem is not related to a position effect. The staining of isolated embryos, which allows the visualization of limited localized expression, may allow detection of expression that is not detected above background in analyses of protein or total RNA prepared from bulk seed samples, the assays used in the other studies. Therefore, the early timing and endosperm expression that we observe, which differ from the endogenous gene expression in soybean seed as determined by in situ hybridization, may result from expression of the a’ P-conglycinin promoter in a heterologous host or from a more sensitive assay. The expression we see in the cotyledons and axis of older embryos is in agreement with the expression pattern in maturationstage soybean embryos. Chamberland et al. (1992) also observed uniform staining of the cotyledons and axis of mature embryos bearing a GUS gene expressed from the a’ Pconglycinin promoter. They did not determine GUS localiza- 455 tion in earlier-stage embryos. They describe a light staining of the endosperm in mature seed, which indicates lower expression than we observe in that tissue. The promoter of the French bean P-phaseolin gene has also been reported to be expressed in mid- to late-maturation stages (Sun et al., 1978; Murray and Kennard, 1984). In transgenic tobacco seeds analyzed as bulk samples, phaseolin protein (Sengupta-Gopalan et al., 1985) and P-phaseolin promoter-expressed mRNA (Riggs et al., 1989) or marker gene activity (Bustos et al., 1989)were first detected at a midmaturation stage, Deletion analysis of this promoter in transgenic tobacco revealed a complex set of regulatory regions within the sequence between +20 and -795 (Bustos et al., 1991). Since our promoter fragment extends only to -410, it retains only a subset of these domains. It does include upstream activating sequence 1 and lack negative regulatory sequence 2, a combination that results in expression 3 d earlier than that directed by the full-length promoter region. Thus, the shorter length of our &phaseolin promoter may explain the early expression that we observe in heart-stage embryos. However, we do not lose expression in the hypocotyl region of mature embryos, as was reported by Bustos et al. (1991) for a -418 promoter. In their report, longer promoters were expressed throughout the embryo except in the root meristem and cap. All promoter fragments analyzed directed expression in the endosperm as well, in agreement with our observations of endosperm expression. The pattern of expression directed by the a‘ 8-conglycinin and @-phaseolinpromoters in tobacco seed was very consistent among independent transformants in this study. Others have also observed a relative insensitivity to position effects of seed-specific promoters (Chen et al., 1986). Therefore, we assume that the pattern of Cre expression directed by the a’ B-conglycinin and P-phaseolin promoters is similar to the pattem of GUS expression from the same promoters, initiating at a heart stage and extending throughout the embryo in a young torpedo stage. Cre expressed in this manner is able to activate the lox2-GUSgene throughout the entire embryo and endosperm. The pattem of activation in the embryo follows the pattern of promoter activity. Spots of activated GUS activity are first detected at the junction between the cotyledons and embryo axis, the site of highest seed promoter expression. A direct correlation between amount of Cre protein and frequency of recombination was previously established (Sauer and Henderson, 1988). Blue spots are first seen in torpedo-stage embryos, about 1 d in development after the a’ P-conglycinin and P-phaseolin promoters are first active, at the heart stage. This then is the period of time required for Cre protein to be synthesized, enter the nucleus, identify lox sites within the plant genome, and recombine these sites, producing a functional gene. Entering the nucleus does not seem to be a limiting factor, since the addition of a nuclear-targeting signal to Cre did not improve the timing of lox2-GUS activation. Cre is a small, 38-kD protein that should be able to pass through the nuclear pore complex (Garcia-Bustos et al., 1991). However, small nuclear-localized proteins do contain localization signals to facilitate their entry (Raikhel, 1992). The SV40 large Tantigen nuclear localization signal did effectively direct the localization of T7 polymerase to the plant cell nucleus (Lass- Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. 456 Odell et al. ner et al., 1991). Since we did not directly assay the effect of this signal on nuclear localization of Cre or on Cre enzymatic activity, it is possible that reduction in recombination rate and enhancement from nuclear targeting could have been balanced, opposing effects. In any case, our attempt to reduce the time period required for gene activation by addition of the SV40 large T-antigen nuclear localization signal to Cre was not successful. Rapid expression of the lox2-GUS gene was achieved by directing recombination prior to the time when the seedspecific promoter was initially active. The GUS gene was then already in the active state when expression of its seedspecific promoter initiated, eliminating the delay in production of GUS enzyme activity. Cre expressed from the early, highly active 355 promoter/Cab leader directs this preactivation. Earlier expression of the 35s promoter than the a‘ pconglycinin promoter was inferred previously in a study on seed-specific inhibition of GUS expression, since GUS regulated by a 35s promoter was expressed, then shut off by an antisense RNA expressed from the a’ 8-conglycinin promoter (Fujiwara et al., 1992). Expression of the 35s promoter by 10 dap was also discussed by Scofield et al. (1993). By staining for GUS activity in dissected embryos we were able to determine that the 35SC regulatory sequence is active in heartstage embryos but not in globular embryos. Since the 35s promoter is active in many tissues and cell types (Nagy et al., 1985; Odell et al., 1985; Jefferson et al., 1987), its inactivity in globular embryos should be noted. Attempts to complement a globular-embryo-stage mutation by expressing the wild-type coding region under control of the 35s promoter would not be effective. Inactivity in the globular embryo might be explained by the modular nature of the 35s promoter. It is composed of domains and subdomains that confer different tissue-specific and developmental pattems of expression (Benfey et al., 1989, 1990; Lam et al., 1989). One identified domain confers expression in radicle and root tissue, whereas another confers expression in cotyledons and most seedling tissues. These tissue-specific cis elements may not be recognized by cells of the undifferentiated globular embryo, but by the heart stage, cells of the embryo may have already acquired enough identity with radicle, axis, or cotyledon tissue to express factors that interact with the domains of the 35S/P, allowing its expression. The presence of the activated GUS gene in F2 heart-stage embryos, even when the cre gene is not inherited, indicates that recombination occurred prior to FZ embryo formation. Activation could have occurred in the F1 embryonic shoot meristem or in any tissue of the F1 plant from which genetically effective cells arise. By GUS staining, both the 35s promoter and seed-specific promoters appear to be active in the shoot meristem region of the embryo. Activity of the pphaseolin promoter in the embryonic shoot meristem was seen previously by GUS staining of mature transgenic tobacco seed (Bustos et al., 1991). Seed-specific promoters are not generally known to be active other than in the embryo, in concordance with Cre-mediated GUS gene activation occurring in the embryonic shoot meristem. Recently, however, expression of GUS directed by the p-phaseolin promoter was observed in apical meristems of transgenic tobacco plants (Sen et al., 1993). Plant Physiol. Vcll. 106, 1994 Comparison of the efficiencies of recombinaíion directed by our 35SC-cre and 35s-cre genes supports the importance of the rnRNA sequence in contributing to the lek el of protein expression. Infemng from the timing of lox2-GUSgene activation, the 35SC-cre gene is expressed much more highly than the 35s-cre gene. These genes do have different 3‘ ends: Ocs 3’ versus Nos 3’, which could contribute to mRNA processing and stability. However, there is a striking difference in the sequence surrounding the translati on-initiating ATG, 35SC-cre having an almost perfect consenijus sequence (Kozak, 1986) and 35s-cre having little homology. Thus, mRNA translatability may be the predominant basis for the discrepancy in Cre expression. During these experiments we also saw differences in levels of Cre expression, inferred from the timing of lo.rz-GUSgene activation, among plants belonging to the same 35s-cre line. Several different 35s-cre plants from the Cre3 line were used in cros!jes to activate lox2-GUS. They were all grown from seed produced by selfing an RI plant that was homozygous at a single locus for the linked selection marker Thus, these plants were expected to be genetically identical. ‘Twoof them directed the same pattem of lox2-GUS-activatedGUS activity in F1 seed as expected, whereas one directed much less activation. Reduced Cre activity in this plant, as compared to its siblings, could be due to methylation of the 35s-cre transgene. Variable loss of transgene expressiori among siblings has been correlated with methylation in some studies (Lin et al., 1990; Kilby et al., 1992; Asaad et al., 1993). We have not compared the methylation of the 3551-cregene in this plant and its siblings. The reduced recombination directed by this 35s-cre plant, and also by the plants of the Cre2 line, pr0duc.s a spotting pattem on maturing cotyledon-stage embryos. Small spots indicate sites of recent lox2-GUS activation, whereas larger spots indicate an earlier recombination event and include cells descending from the one in which loxZ-GIJSwas activated. 1,imited recombination provides a system in which cell lineages may be followed. Limited recombinabon could be directed by low expression of Cre in any tissue, producing a mosaic pattern of expression of a target gene. This system could be used to restore function of a wild-type gene in the corresponding mutant background, in a cell-localized manner. Effects of cell-autonomous mutant restorohon on the tissue would indicate whether a cell’s response to the gene product is transmitted among the cells of the tissue or remains limited to the cell in which it is expressed. This study on tissue-specific Cre-mediated activation of a GUS marker gene, whose expression is initially blocked by a lox-bounded fragment, provides a basis for applying the Cr ./lox recombination system to these types of developmenta I studies. ACKNOWLEDGMENTS We wish to thank C. Patterson for generating transformed tobacco plants, :H. Hershey and E. Heppard for preparing the cre coding region with the 5’ NcoI site, C. Wandelt for the modified plasmids containing the seed promoters and for the nuclear localization signal, M. Locke for the binary vector pZS198, K. Reiter for help with chemiluminescent Southem blots, and J. Slightom for the 0-phaseolin and a’ j3-conglycinin promoters. We also thank S . Russell and J. Downloaded from on August 3, 2017 - Published by www.plantphysiol.org Copyright © 1994 American Society of Plant Biologists. All rights reserved. Crellox-Mediated Seed-Specific Gene Activation Shen for valuable discussions and E. Krebbers for critical reading of the manuscript. Received April 14, 1994; accepted June 28, 1994. Copyright Clearance Center: 0032-0889/94/106/0447/12. LITERATURE CITED An G, Ebert PR, Mitra A, Ha SB (1988) Binary vectors. In SB Gelvin, RA Schilperoort, eds, Plant Molecular Biology Manual. Kluwer Academic Publishers, Dordrecht, The Netherlands, pp A31-19 Assaad FF, Tucker KL, Signer ER (1993) Epigenetic repeat-induced gene silencing (RIGS) in Arabidopsis. Plant Mol Biol22 1067-1085 Bayley CC, Morgan M, Dale EC, Ow DW (1992) Exchange of gene activity in transgenic plants catalyzed by the Cre-lox site-specific recombination system. 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