A new approach for the identification and cloning of genes: the pBACwich system using Cre/lox site-specific recombination

Abstract

With current plant transformation methods ( Agrobacterium, biolistics and protoplast fusion), insertion of DNA into the genome occurs randomly and in many instances at multiple sites. Associated position effects, copy number differences and multigene interactions can make gene expression experiments difficult to interpret and plant phenotypes less predictable. An alternative approach to random integration of large DNA fragments into plants is to utilize one of several site-specific recombination (SSR) systems, such as Cre/ lox. Cre has been shown in numerous instances to mediate lox site-specific recombination in animal and plant cells. By incorporating the Cre/ lox SSR system into a bacterial artificial chromosome (BAC) vector, a more precise evaluation of large DNA inserts for genetic complementation should be possible. Site-specific insertion of DNA into predefined sites in the genome may eliminate unwanted 'position effects' caused by the random integration of exogenously introduced DNA. In an effort to make the Cre/ lox system an effective tool for site-directed integration of large DNAs, we constructed and tested a new vector potentially capable of integrating large DNA inserts into plant and fungal genomes. In this study, we present the construction of a new BAC vector, pBACwich, for the system and the use of this vector to demonstrate SSR of large DNA inserts (up to 230 kb) into plant and fungal genomes.

Figure 1

Construction of pBACwich. pBACKAN was modified by inserting a DNA fragment that contains a lox site, a promoterless hpt (hygromycin phosphotransferase coding region) gene and the nos 3′ polyadenylation sequence (lox–hpt–nos 3′). The DNA fragment from plasmid p24 (Dr D. Ow, Plant Gene Expression Center) was ligated on the 3′-end at the NotI restriction site. At the 5′-end, the fragment was inserted by site-specific recombination in vitro at the lox site already present on the BAC vector using Cre recombinase. After construction, all of the cloning junctions were verified by DNA sequencing.

Figure 2

Diagram of the pBACwich vector. The plasmid is based on pBeloBAC11. CM^R, chloramphenicol resistance; 35S, 35S dual enhancer promoter; KAN^R, kanamycin resistance; hpt, hygromycin resistance. 35S and KAN^R can be used for random transformation in plants and hpt can be used for site-specific integration in plants or animals.

Figure 3

Construction of pSD. Details are provided in Materials and Methods.

Figure 4

The structure of plasmid pSD to create a lox site in M.grisea. Pgpd, A.nidulans gpdA (glyceraldehyde-3-phosphate dehydrogenase) promoter; lox76, 5′-ATAACTTCGTATAGCATACATTATACGcccggta-3′; cre, Cre recombinase; hpt, hygromycin phosphotransferase; TtrpC, A.nidulans trpC terminator; AMP^R, ampicillin resistance; BAR, Basta resistance.

Figure 5

Experimental design of Cre-mediated site-specific recombination. PCR primers (s, h, w and c) are shown as triangles, with the numbers indicating the sizes in kb of the expected PCR products and the DNA fragments after digestion with EcoRI (E) and BamHI (B).

Figure 6

PCR analysis and restriction analysis of the PCR products. A specific band (1.1 kb) was amplified using primers s and c (sc) from the recipient plant (1999.5, harboring a single 35S–loxP–npt construct) but not from the pBACwich150-transformed plants (tobacco 3, 4 and 6) (see Fig. 5). All primers (s, c, h and w) were used in lane a for each DNA to see if any fragments would be amplified by any combinations. Two bands, obtained with primers s and h (sh, 0.61 kb) and w and c (wc, 1.37 kb), were amplified from pBACwich150-transformed tobacco but not from the recipient tobacco. BamHI cleaved the 1.1 kb sc product from the recipient into three fragments of 0.02, 0.41 and 0.67 kb (b in recipient). The 0.61 kb sh product from the pBACwich150-transformed plant was cleaved by EcoRI into two fragments of 0.35 and 0.26 kb (e in tobacco 3, 4 and 6). The 1.37 kb wc product from the pBACwich150-transformed tobacco was also cleaved by BamHI into two fragments of 0.7 and 0.67 kb (b in tobacco 3, 4 and 6).

Figure 7

Southern blot analysis of tobacco genomic DNA from R₀ tobacco plants transformed with pBACwich150. All samples were digested with HindIII. (A) The restriction fragments hybridized with cre probe were shifted in all transformants, indicating DNA rearrangement (see Fig. 5). (B) The hpt probe hybridized to transformants that included the hpt gene but not to the recipient. The recipient tobacco is referred to as 1999.5, and 3, 12, 4, A and B refer to transformed tobaccos with pBACwich150.

Figure 8

Analysis of tobacco genomic DNA from R₀ tobacco plants transformed with the cotton pBACwich150 insert. (A) All samples were digested with HindIII and hybridized with the left forward DOP PCR product of pBACwich150. (B) All samples were digested with HindIII and EcoRV and hybridized with the right backward DOP PCR product of pBACwich150. 1999.5, recipient; 3, 12, 4, A and B, transformants.