Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations

The efficient production of stable transgenic plants is important for both crop improvement and functional genomics. Site-specific integration of foreign genes into a designated genomic position is an attractive tool for minimizing expression variability between transgenic lines. Here, we studied the utility of a Cre-mediated, site-specific integration approach, facilitated by particle bombardment, for streamlining the production of stable transgenic plants, using rice as a model species. Using this method, we generated 18 different transgenic lines containing a precise integration of a single copy of beta-glucuronidase gene (gusA) into a designated genomic location. Eleven of these lines contained no illegitimate integration in the background (single-copy lines), and seven contained illegitimate integrations in addition to the site-specific integration (multicopy lines). We monitored gusA expression in these lines up to three to four successive generations. Each of the single-copy lines expressed the gusA gene at consistent levels and nearly doubled the expression level in the homozygous state. In contrast, multicopy lines displayed expression variation and gene silencing. In about half of the multicopy lines, however, expression of the site-specific integration locus could be reactivated and stabilized on segregation of the illegitimate integrations, whereas, in the remaining half, expression could not be restored, as they contained genetically linked illegitimate integrations. This study demonstrates that biolistic-mediated, site-specific gene integration is an efficient and reliable tool for streamlining the production of stable transgenic plants.     

Site-specific gene integration in rice genome mediated by the FLP-FRT recombination system

Plant transformation based on random integration of foreign DNA often generates complex integration structures. Precision in the integration process is necessary to ensure the formation of full-length, single-copy integration. Site-specific recombination systems are versatile tools for precise genomic manipulations such as DNA excision, inversion or integration. The yeast FLP-FRT recombination system has been widely used for DNA excision in higher plants. Here, we report the use of FLP-FRT system for efficient targeting of foreign gene into the engineered genomic site in rice. The transgene vector containing a pair of directly oriented FRT sites was introduced by particle bombardment into the cells containing the target locus. FLP activity generated by the co-bombarded FLP gene efficiently separated the transgene construct from the vector-backbone and integrated the backbone-free construct into the target site. Strong FLP activity, derived from the enhanced FLP protein, FLPe, was important for the successful site-specific integration (SSI). The majority of the transgenic events contained a precise integration and expressed the transgene. Interestingly, each transgenic event lacked the co-bombarded FLPe gene, suggesting reversion of the integration structure in the presence of the constitutive FLPe expression. Progeny of the precise transgenic lines inherited the stable SSI locus and expressed the transgene. This work demonstrates the application of FLP-FRT system for site-specific gene integration in plants using rice as a model.     

Dosage-Dependent Gene Expression from Direct Repeat Locus in Rice Developed by Site-Specific Gene Integration

In the standard plant transformation practice, transgene copy number is often inversely correlated with transgene expression. As the integration locus generated by standard methods is mostly complex, consisting of both full-length and partial copies arranged in direct or inverted repeat configurations, it is difficult to parse the effect of copy number and locus structure. To clearly study the effect of transgene copy number on gene expression, it is important to control the locus structure and integrate full-length copies. In this study, the effect of transgene copy number on transgene expression in plant cells was determined using rice callus as a model. To generate full-length integrations, Cre-lox-mediated site-specific gene integration method was used. Transgenic rice lines consisting of one to three copies of β-glucuronidase or green fluorescent protein genes were developed. Site-specific integration lines were characterized and subjected to expression analysis. Lines containing two or three copies of either reporter genes displayed 2–4 times higher expression compared to the single-copy lines. Therefore, dosage-dependent transgene expression can be obtained by integrating full-length copies, and site-specific gene integration approach can serve as an efficient tool for generating precise multi-copy integrations.     

FLP-mediated recombination for use in hybrid plant production

We have studied the feasibility in Arabidopsis of using a site-specific recombination system FLP/FRT, from the 2 microm plasmid of yeast, for making plant hybrids. Initially, Arabidopsis plants expressing the FLP site-specific recombinase were crossed with plants transformed with a vector containing kanamycin-resistance gene (npt) flanked by FRT sites, which also served to separate the CaMV35S promoter from a promoterless gusA. Hybrid progeny were tested for excision of the npt gene and the positioning of 35S promoter proximal to gusA. GUS activity was observed in the progeny of all crosses, but not in the progeny derived from the self-pollinated homozygous parents. We then induced male sterility in Arabidopsis plants using the antisense expression of a pollen- and tapetum-specific gene, bcp1, flanked by FRT sites. Upon cross-pollination of flowers on the same male-sterile plants with pollen from FLP-containing plants, viable seeds were produced and the progeny hybrid plants developed normally. Molecular analyses revealed that the antisense expression cassette of bcp1 had been excised in these plants. These results show for the first time that a site-specific recombinase can be used to restore fertility in male-sterile plants, providing an alternative method for the production of hybrid seeds and plants.     

Expression of a transgene exchanged by the recombinase-mediated cassette exchange (RMCE) method in plants

We developed a site-directed integration (SDI) system for Agrobacterium-mediated transformation to precisely integrate a single copy of a desired gene into a predefined target locus by recombinase-mediated cassette exchange (RMCE). We produced site-specific transgenic tobacco plants from four target lines and examined expression of the transgene in T1 site-specific transgenic tobacco plants, which were obtained by backcrossing. We found that site-specific transgenic plants from the same target lines showed approximately the same level of expression of the transgene. Moreover, we demonstrated that site-specific transgenic plants showed much less variability of transgene expression than random-integration transgenic plants. Interestingly, transgenes in the same direction at the same target locus showed the same level of activity, but transgenes in different directions showed different levels of activity. The expression levels of transgene did not correlate with those of the target gene. Our results showed that the SDI system could benefit the precise comparisons between different gene constructs, the characterization of different chromosomal regions and the cost-effective screening of reliable transgenic plants.     

Marker-free site-specific gene integration in rice based on the use of two recombination systems

Transgene integration mediated by heterologous site-specific recombination (SSR) systems into the dedicated genomic sites has been demonstrated in a few different plant species. This approach of plant transformation generates a precise site-specific integration (SSI) structure consisting of a single copy of the transgene construct. As a result, stable transgene expression correlated with promoter strength and gene copy number is observed among independent transgenic lines and faithfully transmitted through subsequent generations. Site-specific integration approaches use selectable marker genes, removal of which is necessary for the implementation of this approach as a biotechnology application. As SSR systems are also excellent tools for excising marker genes from transgene locus, a molecular strategy involving gene integration followed by marker excision, each mediated by a distinct recombination system, was earlier proposed. Experimental validation of this approach is the focus of this work. Using FLPe-FRT system for site-specific gene integration and heat-inducible Cre-lox for marker gene excision, marker-free SSI lines were developed in the first generation itself. More importantly, progeny derived from these lines inherited the marker-free locus, indicating efficient germinal transmission. Finally, as the transgene expression from SSI locus was not altered upon marker excision, this method is suitable for streamlining the production of marker-free SSI lines.     

Efficient system for directed integration into the Lactobacillus acidophilus and Lactobacillus gasseri chromosomes via homologous recombination

An efficient method is described for the generation of site-specific chromosomal integrations in Lactobacillus acidophilus and Lactobacillus gasseri. The strategy is an adaptation of the lactococcal pORI system (K. Leenhouts, G. Venema, and J. Kok, Methods Cell Sci. 20:35-50, 1998) and relies on the simultaneous use of two plasmids. The functionality of the integration strategy was demonstated by the insertional inactivation of the Lactobacillus acidophilus NCFM lacL gene encoding beta-galactosidase and of the Lactobacillus gasseri ADH gusA gene encoding beta-glucuronidase.     

FLP-mediated recombination of FRT sites in the maize genome.

Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment. Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place. The functional FLP gene could be either expressed transiently or after stable integration into the maize genome. The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required. The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA.     

Transgene behaviour across two generations in a large random population of transgenic rice plants produced by particle bombardment

The relationship between transgene copy number, rearrangement levels, inheritance patterns, expression levels, transgene stability and plant fertility was analysed in a random population of 95 independently transformed rice plant lines. This analysis has been conducted for both the selectable marker gene ( aphIV) and the unselected reporter gene ( gusA), in the presence or absence of flanking Matrix Attachment Regions (MARs) in order to develop a better understanding of transgene behaviour in a population of transgenic rice plants created by particle bombardment. In the first generation (T(0)), all the independently transformed plant lines contained and expressed the aphIV gene conferring resistance to hygromycin, but only 87% of the lines were co-transformed with the unselected gusA marker gene. Both transgenes seemed to be expressed independently. Most lines exhibited complex transgene rearrangements as well as an intact transgene expression unit for both aphIV and gusA transgenes. Transgene copy number was proportional to the quantity of DNA used during bombardment. In T(0) plants, high gusA copy number significantly decreased GUS expression levels but there was no correlation between expression level and transgene copy number across the entire population of lines. Four main factors impaired transgene expression in primary transgenic plants (T(0)) and their progeny (T(1)): (1) absence of transgene expression in T(0) plants (41% of lines), (2) sterility of T(0) plants (28% of lines), (3) non-transmission of intact transgenes to some or all progenies (at least 14% of lines), and (4) silencing of transgene expression in progeny plants (10% of lines). Transgene stability was significantly related to differences in transgene structure and expression levels. The presence of Rb7 MARs flanking the gusA expression unit had no effect on plant fertility or non-transmission of transgenes, but provided copy number-dependent expression of the transgene and improved expression levels and stability over two generations. Overall, only 7% of the plant lines without MARs and 17% of the lines with MARs initially generated, exhibited stable transgene expression over two generations.     

Heat-inducible expression of FLP gene in maize cells

The soybean heat-shock gene promoter ( Gmhsp 17.5-E ) has been used to direct expression of gusA and FLP genes in maize cells. At inducible temperatures, in transient expression assays, gusA gene expression controlled by the heat-shock promoter is about 10-fold higher than the expression directed by the CaMV 35S promoter. The Gmhsp 17.5-E promoter preserves its regulatory functions in heterologous maize cells after random integration into genomic DNA.
Heat-shock inducible expression of the FLP gene was investigated by co-transformation of the FLP expression vector (pHsFLP) and a recombination test vector (pUFNeo-FmG) into maize protoplasts. Co-transformed protoplasts were incubated at 42°C for 2 h. This treatment induced recombination of 20–25% of the available FRT sites in transient assays. As a result of heat-shock treatment of stably co-transformed maize cells, activation of gusA gene expression and an associated decrease or elimination of NPT-II activity in transgenic maize lines was observed. Molecular evidence was obtained of the expected DNA excision process catalyzed by the FLP protein in maize transgenic cells. Thus, the experiments presented in this paper indicate that the FLP protein can recognize and subsequently recombine the FRT target sites that had integrated into plant genomic DNA, and that regulated expression of the FLP gene is possible in maize cells using the soybean heat-shock promoter.     

FLP recombinase-mediated site-specific recombination in rice

The feasibility of using the FLP/ FRT site-specific recombination system in rice for genome engineering was evaluated. Transgenic rice plants expressing the FLP recombinase were crossed with plants harbouring the kanamycin resistance gene ( neomycin phosphotransferase II , nptII ) flanked by FRT sites, which also served to separate the corn ubiquitin promoter from a promoterless gusA . Hybrid progeny were tested for excision of the nptII gene and the positioning of the ubiquitin promoter proximal to gusA . While the hybrid progeny from various crosses exhibited β-glucuronidase (GUS) expression, the progeny of selfed parental rice plants did not show detectable GUS activity. Despite the variable GUS expression and incomplete recombination displayed in hybrids from some crosses, uniform GUS staining and complete recombination were observed in hybrids from other crosses. The recombined locus was shown to be stably inherited by the progeny. These data demonstrate the operation of FLP recombinase in catalysing excisional DNA recombination in rice, and confirm that the FLP/ FRT recombination system functions effectively in the cereal crop rice. Transgenic rice lines expressing active FLP recombinase generated in this study provide foundational stock material, thus facilitating the future application and development of the FLP/ FRT system in rice genetic improvement.     

Integration,expression and inheritance of two linked T-DNA marker genes in transgenic lettuce

T-DNA integration and stability were assessed in Agrobacterium-derived transgenic lettuce lines carrying a chimaeric CaMV 35S promoter-driven gus-intron gene and a chimaeric nos.nptII.nos gene. T-DNA integration was predominantly complex in transgenic plants derived from an A. tumefaciens strain carrying the supervirulent plasmid ToK47. Truncation of the right side of the T-DNA was observed in first seed generation R₁ plants from one line. Complex T-DNA integration patterns did not always correlate with low transgene expression. Despite a high T-DNA copy number, ca. 30% of the lines analysed showed high transgene expression in the R₁ generation. High transgene expression was stable at least to the R₄ seed generation in selected high-expressing lines. Transgene expression was lost in the R₂ generation in a low expressing line, while complete, heritable transgene silencing from the R₀ to R₂ generations was also observed in another line. A 50-fold variation in -glucuronidase (GUS) activity and a 16-fold variation in NPTII protein content were observed between R₁ plants derived from different R₀ parents. Reactivation of transgene expression with 5-azacytidine in partially silenced lines indicated that low expression was associated with DNA methylation.     

Site-specific deletions in the tomato genome by the CinH-<Emphasis Type="Italic">RS2</Emphasis> and ParA-<Emphasis Type="Italic">MRS</Emphasis> recombination systems

We have tested the CinH-RS2 and ParA-MRS site-specific deletion systems in tomato (Solanum lycopersicum L.). The ParA-MRS system is derived from the broad-host-range plasmid RK2, where the 222 aa ParA recombinase recognizes a 133 bp multimer resolution site (MRS). The CinH-RS2 system is derived from Acinetobacter plasmids pKLH2 and pKLH204, where the 188 amino acid CinH recombinase recognizes a 113-bp recombination site known as RS2. In this study, target lines containing a DNA segment flanked by recombination sites were crossed to recombinase-expressing lines producing CinH or ParA recombinase. CinH-mediated recombination of RS2 substrates was detected in 2 of 3 F₁ plants that harbor both the target and recombinase loci. On the other hand, recombination mediated by ParA was not detected among F₁ plants, but was found among 13 of 47 F₂ plants. These data show that both systems can mediate site-specific DNA deletion in the tomato genome, and, upon further refinement, can provide additional molecular tools for tomato improvement through precise genome manipulation. As the target construct also contains additional recombination sites for site-specific integration by other recombination systems, these tomato lines could be used for future testing of gene stacking through site-specific integration.     

Integration specificity of phage phiC31 integrase in the human genome

The site-specific integrase from bacteriophage phiC31 functions in mammalian cells and is being applied for genetic engineering, including gene therapy. The phiC31 integrase catalyzes precise, unidirectional recombination between its 30-40-bp attP and attB recognition sites. In mammalian cells, the enzyme also mediates integration of plasmids bearing attB into native sequences that have partial sequence identity with attP, termed pseudo attP sites. Here, we analyzed the features of phiC31-mediated integration into pseudo attP sites in the human genome. Sequence analysis of 196 independent integration events derived from three cell lines revealed approximately 101 integration sites: 56% of the events were recurrent integrations distributed among 19 pseudo attP sequences. Bioinformatics analysis revealed a approximately 30-bp palindromic consensus sequence motif shared by all of the repeat occurrences and most of the single occurrence sites, verifying that phiC31-mediated integration into pseudo attP sites is significantly guided by DNA sequence recognition. The most favored unique sequence in these cell lines occurred at chromosome 19q13.31 and accounted for 7.5% of integration events. Other frequent integration sites were in three specific sequences in subfamilies of ERVL and L1 repetitive sequences, accounting for an additional 17.9% of integration events. Integrations could occur in either orientation at a pseudo attP site, were often accompanied by small deletions, and typically occurred in a single copy per cell. A number of aberrant events were also described, including large deletions and chromosome rearrangements. phiC31 integrase-mediated integration only slightly favored genes and did not favor promoter regions. Gene density and expression studies suggested chromatin context effects. An analysis of the safety of integration sites in terms of proximity to cancer genes suggested minimal cancer risk. We conclude that integration systems derived from phiC31 integrase have great potential utility.     

Site-specific gene integration technologies for crop improvement

Targeted integration of foreign genes into plant genomes is a much sought-after technology for engineering precise integration structures. Homologous recombination-mediated targeted integration into native genomic sites remained somewhat elusive until made possible by zinc finger nuclease-mediated double-stranded breaks. In the meantime, an alternative approach based on the use of site-specific recombination systems has been developed which enables integration into previously engineered genomic sites (site-specific integration). Follow-up studies have validated the efficacy of the site-specific integration technology in generating transgenic events with a predictable range and stability of expression through successive generations, which are critical features of reliable and practically useful transgenic lines. Any DNA delivery methods can be used for site-specific integration; however, best efficiency is mostly obtained with direct DNA delivery methods such as particle bombardment. Although site-specific integration approach provides unique advantages for producing transgenic plants, it is still not a commonly used method. The present article discusses barriers and solutions for making it readily available to both academic research and applicative use.