Site-Specific Integration of Transgenes in Soybean via Recombinase-Mediated DNA Cassette Exchange

A targeting method to insert genes at a previously characterized genetic locus to make plant transformation and transgene expression predictable is highly desirable for plant biotechnology. We report the successful targeting of transgenes to predefined soybean (Glycine max) genome sites using the yeast FLP-FRT recombination system. First, a target DNA containing a pair of incompatible FRT sites flanking a selection gene was introduced in soybean by standard biolistic transformation. Transgenic events containing a single copy of the target were retransformed with a donor DNA, which contained the same pair of FRT sites flanking a different selection gene, and a FLP expression DNA. Precise DNA cassette exchange was achieved between the target and donor DNA via recombinase-mediated cassette exchange, so that the donor DNA was introduced at the locus previously occupied by the target DNA. The introduced donor genes expressed normally and segregated according to Mendelian laws.Plant transformation has challenges such as random integration, multiple transgene copies, and unpredictable expression. Homologous recombination (Iida and Terada, 2005; Wright et al., 2005) and DNA recombinase-mediated site-specific integration (SSI) are promising technologies to address the challenges for placing a single copy of transgenes into a precharacterized site in a plant genome.Several site-specific DNA recombination systems, such as the bacteriophage Cre-lox and the yeast FLP-FRT and R-RS, have been used in SSI studies (Ow, 2002; Groth and Calos, 2003). A common feature of these systems is that each system consists of a recombinase Cre, FLP, or R and two identical or similar palindromic recognition sites, lox, FRT, or RS. Each recognition site contains a short asymmetric spacer sequence where DNA strand exchange takes place, flanked by inverted repeat sequences where the corresponding recombinase specifically binds. If two recognition sites are located in cis on a DNA molecule, the DNA segment can be excised if flanked by two directionally oriented sites or inverted if flanked by two oppositely oriented sites. If two recognition sites are located in trans on two different DNA molecules, a reciprocal translocation can happen between the two DNA molecules or the two molecules can integrate if at least one of them is a circular DNA (Ow, 2002; Groth and Calos, 2003).Single-site SSI can integrate a circular donor DNA containing one recognition site into a similar site previously placed in a plant genome. The integrated transgene now flanked by two recognition sites is vulnerable to excision. Transient Cre expression and the use of mutant lox sites to create two less compatible sites after integration helped reduce the subsequent excision in tobacco (Nicotiana tabacum; Albert et al., 1995; Day et al., 2000). A similar approach was used to produce SSI events in rice (Oryza sativa), and the transgene was proven stably expressed over generations (Srivastava and Ow, 2001; Srivastava et al., 2004; Chawla et al., 2006). Using a promoter trap to displace a cre gene in the genome with a selection gene from the donor, approximately 2% SSI was achieved in Arabidopsis (Arabidopsis thaliana; Vergunst et al., 1998).When two recognition sites located on a linear DNA molecule are similar enough to be recognized by the same recombinase but different enough to reduce or prevent DNA recombination from happening between them, the DNA segment between the two sites may not be easily excised or inverted. When a circular DNA molecule carrying the same two incompatible sites is introduced, the circular DNA can integrate by the corresponding recombinase at either site on the linear DNA to create a collinear DNA with four recognition sites, two from the original linear DNA and two from the circular DNA. DNA excision can subsequently occur between any pair of compatible sites to restore the two original DNA molecules or to exchange the intervening DNA segments between the two DNA molecules. This process, termed recombinase-mediated cassette exchange (RMCE), can be employed to integrate transgenes directionally into predefined genome sites (Trinh and Morrison, 2000; Baer and Bode, 2001).RMCE using two oppositely oriented identical RS sites, a donor containing the R recombinase gene and a third RS site to limit random integration, resulted in cassette exchange between the donor and a previously placed target in tobacco (Nanto et al., 2005). RMCE using both the Cre-lox and FLP-FRT systems improved RMCE frequency in animal cell cultures (Lauth et al., 2002). RMCE using two directly oriented incompatible FRT sites and transiently expressed FLP recombinase achieved cassette exchange between a target previously placed in the Drosophila genome and a donor introduced as a circular DNA (Horn and Handler, 2005). A gene conversion approach involving Cre-lox- and FLP-FRT-mediated SSI, RMCE, and homologous recombination was explored in maize (Zea mays; Djukanovic et al., 2006). RMCE using two oppositely oriented incompatible lox sites and transiently expressed Cre recombinase produced single-copy RMCE plants in Arabidopsis (Louwerse et al., 2007).To develop FLP-FRT-mediated RMCE in soybean (Glycine max), we created transgenic target lines containing a hygromycin resistance gene flanked by two directly oriented incompatible FRT sites via biolistic transformation. Single-copy target lines were selected and retransformed with a donor DNA containing a chlorsulfuron resistance gene flanked by the same pair of FRT sites. An FLP expression DNA was cobombarded to transiently provide FLP recombinase. RMCE events were obtained from multiple target lines and confirmed by extensive molecular characterization.