Crop Science
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Published online 1 February 2006
Published in Crop Sci 46:700-705 (2006)
© 2006 Crop Science Society of America
677 S. Segoe Rd., Madison, WI 53711 USA
This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Agricola
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Related Collections
Right arrow Cell Biology & Molecular Genetics
Right arrow Crop Genetics

GENOMICS, MOLECULAR GENETICS & BIOTECHNOLOGY

Efficient Deletion of Transgenic DNA from Complex Integration Locus of Rice Mediated by Cre/lox Recombination System

Sarah K. Moorea and Vibha Srivastava*,b

a Department of Crop, Soil & Environmental Sciences, University of Arkansas, Fayetteville, AR 72701
b Department of Crop, Soil & Environmental Sciences, and Department of Horticulture, University of Arkansas, Fayetteville, AR 72701

* Corresponding author (vibhas{at}uark.edu)


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS
 REFERENCES
 
Transgenic plants may contain several DNA elements, most notably the selectable marker genes, that are not needed after the selection of the transgenic line. Using site-specific recombination system, such as Cre/lox, targeted deletion of these so-called unneeded DNA elements can be achieved. To study the efficiency of Cre/lox-mediated deletion of transgenic DNA from complex locus generated by particle bombardment, we generated two transgenic rice (Oryza sativa L.) lines that contain several copies of lox-flanked ß-glucuronidase (GUS) gene (gusA) and unmarked hygromycin resistance (hpt) gene and crossed them with a Cre-expressing line. Excision of both gusA and hpt genes occurred in F1 hybrids. Molecular data demonstrated that Cre/lox recombination was initiated randomly in F1 plants leading to complete excision of all transgene fragments in somatic tissue. F2 analysis suggested that efficient excision also occurred in germline, preventing the transfer of lox-flanked genes into most of the F2 plants. Further, the excised DNA did not apparently re-integrate into the genome. Thus, Cre/lox-mediated DNA recombination in rice is highly suitable for the removal of the unneeded transgenic DNA.

Abbreviations: bp, base pair • GUS, ß-glucuronidase • gusA, ß-glucuronidase gene • npt, neomycin phosphotransferase gene • hpt, hygromycin phosphotransferase gene • PCR, polymerase chain reaction


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS
 REFERENCES
 
PRODUCTION of transgenic plants is important for crop improvement. Several transgenic varieties of corn (Zea mays L.), soybean [Glycine max (L.) Merr.], and cotton (Gossypium hirsutum L.) are available in the market. As new trait genes are identified through genome sequencing and functional genomics efforts, efficient transformation methods will be required for the economical production of transgenic varieties (Birch, 1997; Ow, 2002). In general, there are two bottlenecks in generating useful transgenic lines, transformation efficiency and stable transgene expression. Both of these issues have been addressed by numerous studies (reviewed by Lorence and Verpoorte, 2004), which have resulted in significant improvements in transformation efficiencies of important crop species. These studies have also suggested that stable transgene expression is most reliably obtained from single-copy (transgene) locus. One of the most efficient procedures for generating single-copy plants is site-specific integration of foreign genes mediated by heterologous site-specific recombination systems (Albert et al., 1995; Srivastava et al., 2004). A number of site-specific recombination systems are functional in model plant species (Lyznik et al., 1993; Dale and Ow, 1991; Ebinuma et al., 1997), with Cre/lox being the most efficient one so far (Andreas et al., 2002; Radhakrishnan and Srivastava, 2005). Phage P1–derived Cre/lox system consists of a 38.5-kDa recombinase Cre that catalyzes recombination between 34-bp recombination targets called lox sites. Cre-mediated recombination between lox sites may result in deletion of the lox-flanked DNA or integration of DNA circle containing a lox site into chromosomal lox site (see Fig. 1 ). Thus Cre/lox system has been utilized for removing selectable marker genes from transgene locus (Dale and Ow, 1991; Hoa et al., 2002; Russell et al., 1992; Sreekala et al., 2005; Zhang et al., 2003; Zuo et al., 2001), and generating site-specific integration of the transformed DNA (Albert et al., 1995; Srivastava et al., 2004; Vergunst et al., 1998). Although most selectable marker genes pose no apparent threat to environment or health (Miki and McHugh, 2004), their removal is highly desirable to mitigate public concerns over the safety of transgenic plants (FAO/WHO, 2000). Cre-mediated site-specific integration is most efficient when DNA is introduced by particle bombardment (Srivastava et al., 2004). Cre-mediated gene integration using PEG-mediated protoplast transformation or Agrobacterium-mediated transformations, on the other hand, generated high rate of gene silencing or low transformation frequency, respectively (Day et al., 2000; Vergunst et al., 1998). Therefore, to make this technology independent of DNA delivery methods, we investigated whether site-specific integration can be obtained by crossing in donor and target lines; such transgenic lines can be developed by any transformation method. Cre-mediated DNA excision and re-integration (site-specific) would occur in F1 hybrids. In addition, this experimental setup was designed to assess the efficiency of excision of the marked (lox-flanked) gene from complex locus generated by cobombardment of two separate plasmids, and fate of the cotransformed unmarked gene after Cre/lox recombination in the locus. Marker gene removal has so far been studied in transgenic lines generated by Agrobacterium-mediated delivery of a single T-DNA containing gene of interest and lox-flanked marker gene (Dale and Ow, 1991; Hoa et al., 2002; Russell et al., 1992; Sreekala et al., 2005; Zhang et al., 2003; Zuo et al., 2001). In particle bombardment–mediated transformation, often two separate plasmids or DNA molecules (one for gene of interest and one for marker gene) are cobombarded, which frequently integrate into a single genomic locus (Kohli et al., 2003; Pawlowski and Somers, 1998) Therefore, we studied the efficiency of Cre-mediated gene excision from transgene locus generated by cobombardment of two separate plasmids, one containing lox-flanked genes and the other containing an unmarked gene. We also sought to determine the effect of Cre-mediated recombination on the structure of the unmarked gene in the transgene locus. A recent study on marker gene removal from transgenic rice reported highefficiency of Cre-mediated excisions in F1 hybrid plants (Hoa et al., 2002). However, stable inheritance of marker-free locus is crucial for plant breeding. Therefore, we tracked the presence of transgenes in F2 population.


Figure 1
View larger version (17K):
[in this window]
[in a new window]
  		Fig. 1. Schematic representation of DNA constructs, and molecular strategy of Cre/lox-mediated gene deletion and re-integration. Donor locus developed by cobombardment of plasmids, p35Shpt and pVS55, contains loxP- and lox75-flanked neomycin phosphotransferase gene (npt) (promoterless) and ß-glucuronidase gene (gusA). Target locus developed by the integration of pVS52 construct contains a cre transcription unit and a target lox76 site. Cre/lox recombination is expected to excise lox-flanked DNA fragments in the form of extra-chromosomal circles containing lox75 that may interact with the genomic lox76 site in the target T5 locus to generate a predictable integration locus. Lox sites are represented as arrowheads between filled (mutant) or empty (wild-type) bars. EcoRI (E) sites are shown for each vector and expected fragment sizes are given in kb. Position of PCR primers are indicated by arrowheads. DNA fragments used to probe Southern blot are underlined. 35S, CaMV 35S promoter; hpt, hygromycin phosphotransferase gene; nos, nopaline synthase transcription termination signal; pubi, maize ubi-1 promoter; loxP wild-type lox site; lox75, mutant (left arm) lox site; lox76, mutant (right arm) lox site; dmlox, double mutant lox site.

 
The data presented here suggests that (i) high efficiency of somatic and germline excision were mediated by Cre/lox system; (ii) all copies of both lox-flanked gene and unmarked gene may be deleted from complex locus generated by cobombardment of two separate plasmids; (iii) the excised DNA does not apparently re-integrate into the genome. The implications of these findings are discussed here.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS AND DISCUSSIONS
 CONCLUSIONS
 REFERENCES
 
Rice Transformation
Callus induction and proliferation and regeneration were performed as described by Hiei et al. (1994). Scutellar calluses were generated from mature seeds of rice cultivar Nipponbare. Calluses were induced and maintained on 2N6 media (N6 basal salts with N6 vitamins, 1 g L–1 casamino acids, 0.1 g L–1 myo-inositol, 30 g L–1 sucrose, and 2 mg L–1 2,4-dichlorophenoxyacetic acid [2,4-D]) and kept in dark at room temperature. Two-month-old callus explants were bombarded with 1-µm gold particles coated with plasmid DNA using the method described by Sanford et al. (1991). A mixture of 5 µg of pVS55 and 5 µg of p35Shpt was adsorbed on 300 µg of gold particles. About 30 µg of the coated particles were bombarded on each callus plate. The callus was pretreated on 2N6 media containing 0.4 M sorbitol for 2 h. Bombarded callus was proliferated for a week on 2N6 media and then transferred to 2N6 media containing hygromycin (50 mg L–1). Resistant colonies, scored 2 to 4 wk later, were transferred to preregeneration media (N6 basal media with 1 mg L–1 6-benzylaminopurine [BAP], 0.5 mg L–1 {alpha}-naphthaleneacetic acid [NAA] and 0.5 mg L–1 abscisic acid [ABA]) containing hygromycin and kept in dark at 25°C for 7 to 9 d. Finally, the calluses were transferred to the regeneration media (N6 basal media with 3 mg L–1 BAP and 0.5 mg L–1 NAA) without selection and kept in continuous fluorescent light at 25°C. Regenerated shoots were rooted in hormone-free MS media (Murashige and Skoog 1962). Well-rooted plants were transferred to greenhouse.

Molecular Analysis
Genomic DNA from rice leaves was isolated using CTAB method (Murray and Thompson 1980). About 5 µg of genomic DNA was digested with EcoRI and separated on 0.8% agarose gel to prepare a Southern blot, which was hybridized with 32P-labeled DNA probes. Gene copy number presented in Table 1 is based on the number of hybridizing bands obtained on the Southern blots. Polymerase chain reaction (PCR) was performed to detect gusA, cre, and hpt genes using primers described in Table 2. The reaction mixture (50 µL) contained 50 mM KCl, 10 mM Tris-HCl (pH 9), 0.1% Triton X-100, 1.5 mM MgCl2, 0.2 mmol each of dATP, dCTP, dGTP, and dTTP, 0.2 µmol of each primer, 0.2 µg of template DNA, and 1 U Taq polymerase (Promega Inc., Madison, WI). PCR reaction consisted of 40 cycles of 1 min denaturation at 94°C, 1 min annealing at 56°C, and 1 min extension at 72°C followed by final elongation step at 72°C for 15 min.


View this table:
[in this window]
[in a new window]
  		Table 1. ß-Glucuronidase (GUS) activity and Southern analysis on F1 plants.

 

View this table:
[in this window]
[in a new window]
  		Table 2. PCR primers.

 
Germination Assay
Mature seeds were surface sterilized for 30 min using 30% commercial bleach with 0.1% SDS, washed three times with autoclaved water, and dried on a paper towel before plating in magenta boxes containing half-strength MS media supplemented with 100 mg L–1 geneticin (Invitrogen, Carlsbad, CA). The boxes were kept at 25°C in continuous light to allow seeds to germinate.

GUS Histochemical Staining
GUS activity was monitored by histochemical staining with 1 mM X-Gluc (Gold Biotechnologies, St. Louis, MO) as described by Jefferson (1987). Punctured mature seeds (soaked in water for a few hours), 3-d-old seedlings or leaf sections from >3-wk-old plants were submerged in 1 mM X-Gluc solution supplemented with 0.5% Tween-20, vacuum infiltrated for 2 h, and incubated overnight at 37°C. After staining, seedlings and leaves were treated with 70% ethanol to remove chlorophyll.


   RESULTS AND DISCUSSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
CONCLUSIONS
 REFERENCES
 
Experimental Design
The main objective of the experimental design described in Fig. 1 was to study the efficiency of Cre-mediated deletion of lox-flanked genomic fragments and their re-integration into the target lox site (T5 locus). The strategy relies on the use of two different transgenic lines, a target line that contains a target lox76 site and expresses cre gene, and a donor line that contains a DNA construct flanked by lox75 and loxP. This construct consists of a promoterless neomycin phosphotransferase (npt) gene and a ß-glucuronidase (gusA) gene. Crossing of these two transgenic lines should allow Cre/lox recombination to occur in F1 plants. As shown in Fig. 1, two different types of Cre/lox reactions are expected, (i) deletions in the donor locus generating a lox75-containing DNA circle of promoterless npt gene and gusA gene, (ii) subsequent re-integration of the excised circle as a result of the recombination between lox75 and lox76 sites. A recombination between lox75 and lox76 will generate a wild-type loxP and a double mutant (dm) lox sites preventing the reversal of the reaction (Albert et al., 1995; Srivastava and Ow, 2002). The resulting integration structure will transcribe npt gene and confer resistance to geneticin. A well characterized target line, T5, was used for this work. T5 locus contains a cre transcription unit and a target lox76 site (Fig. 1). T5 line was previously developed by Agrobacterium-mediated transformation of rice (cv. T309) with pVS52 (Fig. 1; Srivastava and Ow, 2002), which harbors maize ubiquitin-1 promoter (pubi) driven cre gene and Cauliflower mosaic virus 35S RNA promoter (35S) driven hygromycin phosphotransferase (hpt) gene as a selection marker. In the leader sequence of cre gene, a lox76 site is embedded that serves as the target site. Two lox donor lines, L1 and L2 in Nipponbare background, were generated in the present work by particle bombardment of pVS55 DNA along with p35Shpt DNA (Fig. 1). Plasmid pVS55 contains a lox-flanked construct consisting of a promoterless npt gene and a pubi driven gusA gene. Plasmid p35Shpt contains 35S promoter driven hpt gene. T5 plants were crossed with L1 or L2 plants to generate F1 hybrids. Each of the F1 hybrids was grown and allowed to self-fertilize to collect F2 seeds. F1 and the F2 plants were analyzed using PCR and Southern hybridization to track Cre/lox-mediated gene excision and re-integration.

Description of Parental Lines
Cre-expressing line, T5, has been described earlier (Srivastava and Ow, 2002; Srivastava et al., 2004). T5 locus contains a single-copy of pVS52 T-DNA (Fig. 1; Srivastava and Ow, 2002). lox donor lines, L1 and L2, each contain a complex co-integration locus of gusA and hpt genes. L1 locus contains ~20 copies of gusA gene and ~10 copies of hpt gene. L2 locus contains ~7 copies of gusA gene and 1 to 2 copies of hpt gene (Fig. 2 ). Both lines express GUS activity in seedlings and leaves, but L1 displayed random gusA silencing in endosperm and embryos. T5 plants were crossed with each of the donor lines to generate a total of 14 F1 hybrids, seven each for L1 and L2 parents. F2 seeds harvested from the selected F1 plants were subjected to molecular and GUS expression analysis.


Figure 2
View larger version (126K):
[in this window]
[in a new window]
  		Fig. 2. Southern analysis of F1 hybrids. EcoRI-digested genomic DNA isolated from parental lines (L1, L2, and T5), and 8- to 9-wk-old F1 hybrids were probed on Southern blots with radio-labeled DNA fragments of gusA, hpt, and cre genes shown in Fig. 1. The fragment sizes are given in kb.

 
Cre-Mediated Excision Occurred Randomly in Somatic Tissue
Seeds of F1 hybrids were germinated on half-strength MS media; 2-wk-old seedlings were transplanted into pots and grown in greenhouse. GUS activity in the leaf sections of the F1 hybrids was tested at two different stages (1 wk old and 8 wk old) by submerging leaf cuttings in GUS staining solution and incubating overnight at 37°C. Presence of cre and hpt genes in these F1 plants was confirmed by PCR and Southern analysis (described below). All 1-wk-old samples contained strong GUS activity, similar to the parental L1 and L2 lines, indicating the presence of gusA gene in these samples (Table 1). However, spotty GUS activity was detected in some of the 8-wk-old F1 plants, indicating the initiation of Cre/lox reaction, while the remaining showed no activity (Table 1). Next, Southern analysis was done on genomic DNA isolated from 8- to 9-wk-old F1 plants. Six F1 plants of L1 x T5 cross and seven F1 plants of T5 x L2 cross were analyzed by Southern hybridization. All six F1 plants of L1 x T5 were found to completely lack gusA gene. Only three F1 plants of T5 x L2 cross completely lacked gusA gene, and the remaining four were found to contain 2 to 5 gusA copies (Fig. 2; Table 1). The lack of correlation between Southern and GUS activity data (Table 1) may be attributed to different samples, harvesting time, or developmental stages. Indeed, no GUS activity was detected in late-flowering F1 plants. This data indicates random occurrence of Cre/lox-mediated gusA excision in F1 hybrids. Cre-lox recombination was apparently underway in the various developmental stages of F1 plants, as indicated by the reduction in the gusA copy number and deletion of L1 and L2 locus–derived hpt gene (Fig. 2; Table 1). It is noteworthy that each of the F1 plants of both crosses lost all copies of the unmarked hpt gene, as only the fragments specific to T5 locus were observed in F1 plants (Fig. 2). This was further substantiated by F2 data for hygromycin resistance that suggested the presence of a single hpt gene linked with cre locus (discussed below). All 13 F1 plants contained T5-derived cre and hpt genes (Fig. 2; Table 1). Further, the similarity in gusA hybridization pattern of F1 plants suggested that some genomic fragments of L2 locus are more readily excised than others. The observation that all F1 plants derived from L1 x T5 lacked gusA gene suggests that L1 locus is more amenable to Cre/lox reaction than L2 locus. Though unlikely, this difference may also be attributed to the direction of cross, that is, Cre plant serving as female. Previous studies reported no significant difference in recombination efficiency with reciprocal crosses (Hoa et al., 2002; Zhang et al., 2003).

Taken together, these observations suggest, (i) Cre/lox-mediated excisions of genomic locus occurred randomly in somatic tissue in somewhat late developmental stages; (ii) complexity of locus does not negatively impact its amenability to Cre/lox reaction, as L1 locus consisting of ~20 copies was excised more efficiently than L2 locus, containing about half as many copies.

Unmarked Gene Is Also Deleted from Complex Integration Locus
Two plasmids, pVS55 and p35Shpt, were cobombarded to develop L1 and L2 lines. While lox sites flank gusA gene in pVS55 construct, p35Shpt contains an unmarked hpt gene (see Fig. 1). However, co-integration of the two plasmids may generate an integration locus interspersed with lox sites facilitating the deletion of hpt fragments. Indeed, introduction of Cre activity into L1 and L2 lines by crossing with T5 line resulted in deletion of all copies of the unmarked hpt gene. This may have occurred due to the presence of lox-containing fragments on each side of the hpt fragments. This observation suggests that cotransformation of a gene of interest and a marker gene may result in the loss of all transgenic fragments from a complex locus. Therefore, for marker-free transformation approach, both lox-flanked marker gene and unmarked gene of interest should be cloned into a single transformation vector.

Cre/lox-Mediated Gene Excision is Efficient in Germline
To assess Cre/lox-mediated excisions in F2 generation, 3-wk-old F2 plants were analyzed by PCR and Southern hybridization. F2 seeds were collected from a total of five different F1 parents, three representing L1 x T5 cross and two representing T5 x L2 cross. Three-week-old F2 plants, 33 for L1 cross and 23 L2 cross, were subjected to PCR analysis to track the presence of gusA, cre, and hpt gene. While none of these F2 plants contained gusA gene, cre gene was found to be tightly linked with hpt gene (Table 3). This data suggests that hpt gene was inherited only from T5 locus, suggesting the deletion of hpt gene from L1 and L2 locus. Thus efficient germinal and/or somatic excision occurred in each of the samples. Since somatic excisions were initiated late in F1 plants, we assessed germinal excision by testing GUS activity in very young (3 d old) F2 seedlings. Parental L1, L2, and T5 lines served as controls. Overall ~13% F2 seedlings from different crosses displayed weak spotty GUS activity (Table 3). Three-quarters of the F2 seedlings would have stained positive, if somatic and germinal excisions had failed to occur. Thus, absence of GUS activity in ~87% of very young F2 seedlings indicates the occurrence of germinal excisions in F1 plants. This was further suggested by the lack of GUS activity in the endosperm and embryos of the mature F2 seeds. While strong seed (endosperm + embryo) staining was observed in L2 seeds, only ~9% F2 seeds displayed GUS activity (Table 3). Seed staining was not done for L1 crosses because of apparent gene silencing in endosperm of L1 line. Taken together, these observations suggest that somewhat efficient Cre/lox-mediated germinal excision occurred in F1 plants preventing the transmission of lox-flanked genes into some, if not all, F2 progenies. As shown in Fig. 1, Cre-mediated recombination between lox75 and loxP should generate a loxP "footprint" in the donor locus. Since L1 and L2 loci have not been mapped in the rice genome, it was not possible to track the presence of this footprint.


View this table:
[in this window]
[in a new window]
  		Table 3. ß-Glucuronidase (GUS) activity and PCR analysis on F2 plants.

 
The Excised DNA Apparently Does Not Re-Integrate
One of the objectives of the present experimental design was to test if the excised DNA can re-integrate into a given target site. Previous studies have indicated that the excised DNA circles do not randomly re-integrate into the genome at high rate (Dale and Ow, 1991; Hoa et al., 2002; Russell et al., 1992; Sreekala et al., 2005; Zhang et al., 2003; Zuo et al., 2001). In vast majority of the cases, the excised circle is lost through the cell divisions. However, we sought to determine whether the excised circle would re-integrate into T5 locus as a result of Cre-mediated recombination between lox75 and lox76 sites (see Fig. 1). Cre-mediated excision and re-integration was expected to occur in F1 plants resulting in the formation of a defined integration locus conferring resistance to geneticin (Fig. 1). If re-integration occurred efficiently in germ cells, geneticin resistant F2 progenies would be recovered. About 400 F2 seeds derived from five different crosses were plated on half-strength MS media containing 100 mg L–1 geneticin. While a positive control, S2, germinated and elongated, all F2 seedlings succumbed to geneticin toxicity. Seedlings derived from T5, L1, L2, and nontransformed negative control also turned brown and wilted away in geneticin-containing media. S2 is a site-specific integration line containing the integration structure (in T5 locus) shown in Fig. 1. This observation suggests that re-integration of the excised circle into the target lox76 site apparently does not occur. Further, Southern and PCR data of F1 and F2 plants did not suggest random integration of the excised DNA. Therefore, it can be concluded that in the vast majority of the cases the excised DNA does not re-integrate, randomly or site specifically, into the genome. It is important to note that target locus T5 efficiently recombines with the lox-DNA circles delivered by particle bombardment (Srivastava et al., 2004).


   CONCLUSIONS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS
REFERENCES
 
Significant advances have been made in recent years toward improving transformation efficiencies of crop plants (Lorence and Verpoorte, 2004). For commercial production of transgenic plants, generally a large pool of transgenic lines are produced and subjected to molecular analysis to identify single-copy lines, as these lines tend to be stable over successive generations. Therefore, transformation methods designed to generate high number of single-copy plants are highly desirable. Site-specific integration of foreign gene mediated by Cre/lox system produces precise single-copy integration locus that is stable through successive generations (Day et al., 2000; Srivastava et al., 2004). However, this system is efficient only when foreign DNA is delivered by particle bombardment (Srivastava et al., 2004). Another recent improvement in transgenic technology is the development of strategies for removing marker genes. While several methods are available for generating marker-free transgenic plants, they mostly are inefficient or rely on the use of Agrobacterium-mediated transformation approach (Miki and McHugh, 2004). Some plant species, most notably grasses, are more amenable to transformation by particle bombardment, which generally involves cotransformation of two separate plasmids or DNA molecules, one for the gene of interest and one for the marker gene. Therefore, we were interested in investigating (i) whether Cre-mediated DNA excision from transgene locus developed by cobombardment of two separate plasmids is practical, and (ii) whether an alternative approach for site-specific gene integration can be developed that does not depend on the use of particle bombardment. Toward this, we studied the excision of lox-flanked gusA gene and unmarked hpt gene from two complex loci and re-integration of the excised gusA gene into the target lox76 site in rice genome.

The data presented here shows that the Cre/lox system is efficient for somatic and germinal excisions, and therefore extremely useful for marker gene removal from transgenic rice. The efficiency of germinal excisions may be further increased by using meiotic promoters such as plant homologs of DMC1 and CDC45 genes (Klimyuk and Jones, 1997; Stevens et al., 2004). Next, our data shows that the Cre activity can delete both marked and unmarked genes from complex locus generated by particle bombardment. Therefore, for marker-free strategy, single plasmid containing lox-flanked marker gene and unmarked gene of interest should be used. Single plasmid may also generate complex loci, some of which on Cre/lox recombination may lose extra copies along with the marker gene as demonstrated previously in wheat (Triticum aestivum L.) (Srivastava et al., 1999). Such simplified locus will be suitable for crop breeding. However, single plasmid can also generate complex structures with lox sites on both ends; such structures will not be useful for marker deletion purpose. Finally, our data suggests that the excised DNA circles do not re-integrate site specifically into a given target site. Use of meiotic promoters to control Cre activity may improve the chances of re-integration. Until such improvements are done, particle bombardment–mediated site-specific integration remains the most efficient method for generating precise integration structures.

Received for publication August 28, 2005.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS AND DISCUSSIONS
 CONCLUSIONS
 REFERENCES
 




This article has been cited by other articles:


Home page
 Plant Physiol.Home page
T. Komori, T. Imayama, N. Kato, Y. Ishida, J. Ueki, and T. Komari
Current Status of Binary Vectors and Superbinary Vectors
Plant Physiology, December 1, 2007; 145(4): 1155 - 1160.
[Full Text] [PDF]

Home page
 Plant Physiol.Home page
S. De Buck, I. Peck, C. De Wilde, G. Marjanac, J. Nolf, A. De Paepe, and A. Depicker
Generation of Single-Copy T-DNA Transformants in Arabidopsis by the CRE/loxP Recombination-Mediated Resolution System
Plant Physiology, December 1, 2007; 145(4): 1171 - 1182.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Figures Only
Right arrow Full Text (PDF) Free
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (3)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Right arrow Search for Related Content
PubMed
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Agricola
Right arrow Articles by Moore, S. K.
Right arrow Articles by Srivastava, V.
Related Collections
Right arrow Cell Biology & Molecular Genetics
Right arrow Crop Genetics

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
The SCI Journals Agronomy Journal Vadose Zone Journal
Journal of Natural Resources
and Life Sciences Education		 Soil Science Society of America Journal
Journal of Plant Registrations Journal of
Environmental Quality		 The Plant Genome