Nucleic Acids Research

Figure 1. (A) Schematic representation of homologous recombination between pRRP1S and P1 vector. Hatched circle on top represents the P1 vector pAd10-SacBII. The sacB gene (black box) and BamHI cloning site with a genomic insert are shown. White circle on bottom represents pRRP1S. The relative position of the yeast CEN6/ARS4 and LEU2 genes and the bacterial mini-F factor origin of replication (Mini-F) and chloramphenicol resistance (Cm^r) genes are shown. The relative positions of the unique MluI and SalI sites used to linearize RRP1S are shown. The I-SceI sites located in the polylinker of pClasper and used to cut the insert out of pRRP1S are shown. Gray boxes represent recombinogenic ends homologous to P1 vector regions surrounding the cloning site which were amplified by over-lap PCR. The left recombinogenic end is 646 bp long and is homologous to sequences in the sacB gene 138 bp upstream of the BamHI cloning site. It was amplified by the primers: 5[prime]-TGGATCCGGAGTGGTGTGAATCCGTTAGCGA-3[prime] and 5[prime]-AGAAGTGCAGTCGTAACGCGTCGCCGCACTTATGACTGTCTTC-3[prime]. The rightrecombinogenic end is 652 bp long and is 82 bp downstream of the BamHI cloning site. It was amplified by the primers: 5[prime]-GTCATAAGTGCGGCGACGCGTTACGACTGCACTTCTGGCAGGA-3[prime] and 5[prime]-CAAGCTTTTTACCTGCGCTGTTGTCAGGC-3[prime]. After 10 cycles of amplification, the products were diluted, mixed and amplified for an additional 30 cycles using the left 5[prime] primer and right 3[prime] primer. The amplified ends were TA cloned (Invitrogen), digested with BamHI and HindIII and ligated into the BamHI/HindIII sites in the polylinker of pClasper to create pRRP1. SalI linkers were ligated into the MluI site to create pRRP1S. Homologous recombination between pRRP1S and pAd10-SacBII would result in the sequence between the recombinogenic ends, including any inserts in the BamHI site, being transferred to pRRP1S. The arrowheads indicate primers used to PCR across the recombination junction. Primer 1 is 5[prime]-TCGATCAGACTATCAGCGTGA-3[prime] and is located in pRRP1S, 132 bp from the BamHI polylinker site. Primer 4 is 5[prime]-TTAAAGAACGTGGACTCCAACG-3[prime] and is located in pRRP1S 130 nt from the HindIII polylinker site. Primers 2 and 3 are located in the P1 on either side of the BamHI cloning site, but outside the recombinogenic ends. Primer 2 is 5[prime]-AGCCTTCAACCCAGTCAGCTCCTT-3[prime]. Primer 3 is 5[prime]-AATTCGGGAGGATCGAAACGGCA-3[prime]. (B) PCR of recombinant junctions between pRRP1S and P1 after homologous recombination. Primer pairs used are shown. DNA amplified was either the original P1 clone (P1) or yeast genomic DNA of recombinants (yeast).

We used pRRP1S to clone the inserts from two different P1 clones. One clone, P1-cdx2, contained the mouse Cdx-2 gene. The second, P1-ha11, contained the mouse Hoxa-11 gene. pRRP1S and P1 DNAs were prepared for transformation using the Qiagen-tip 500 protocol for very low copy number plasmids (Qiagen). pRRP1S was then linearized by double digestion at the unique MluI and SalI sites designed between the recombinogenic ends (Fig. 1), purified by agarose gel electrophoresis and the excised band was extracted from the gel using the QiaexII Gel Extraction Kit (Qiagen).

pRRP1S and the P1 clones were co-transformed by spheroplasting into the Saccharomyces cerevisiae strain YPH857 (MATa, ura3-52, lys2-801, ade2-101, his3[Delta]200, trp1[Delta]63, leu2[Delta]1, cyh2^r) (8). Cells were grown overnight, diluted to a density of 5 × 10⁶ cells/ml in 100 ml YEPD media then grown at 30°C to a density 1 × 10⁷ cells/ml. Cells were pelleted at 1500 g then resuspended in 10 ml of 1 M sorbitol, 2.4 mM EDTA and 67 mg DTT and incubated for 15 min at 30°C with shaking. Cells were pelleted at 1500 g and resuspended in 10 ml SCE (1 M sorbitol, 0.1 M sodium citrate, pH 5.8, 0.01 M EDTA). The cells were spheroplasted by the addition of 0.2 ml of [beta]-glucuronidase (Sigma) at 30°C for 10-15 min. Cells were collected by centrifugation at 850 g and washed twice in 10 ml of 1 M sorbitol followed by one wash with 10 ml STC (1 M sorbitol, 0.01 M CaCl₂, 0.01 M Tris, pH 7.4). After centrifugation at 850 g, cells were resuspended in 0.5 ml STC. Aliquots of cells (100 µl) were placed in Eppendorf tubes for transformations. An aliquot of 3 µg of pRRP1S and 2-5 µg of P1 DNA was added to the cells and incubated at room temperature for 15 min. 20% PEG solution (1 ml; 20% PEG 3350, 0.01 M CaCl₂ and 0.01 M Tris, pH 7.4) was added and the cells were incubated for an additional 15 min. Cells were collected by centrifugation at 450 g, resuspended in 150 µl SOS media lacking leucine (8) and incubated at 30°C for 20 min. Cells were mixed with equal volume of SS top agar lacking leucine (8) and plated on SORB plates lacking leucine (8). Colonies were visible after 3-4 days at 30°C.

LEU⁺ yeast colonies were screened for recombination by whole cell PCR (4) using primers to the Cdx-2 or Hoxa-11 gene in the P1 inserts. For the co-transformation with P1-cdx2, we screened 44 LEU⁺ colonies, three of which were Cdx-2 PCR positive. Three of 20 LEU⁺ colonies screened from the P1-ha11 co-transformation were PCR positive for Hoxa-11. Plasmids isolated from PCR negative colonies were always circular pRRP1S, indicating that no recombination had occurred in these colonies (data not shown). We believe that these background colonies are due to either uncut pRRP1S being transformed into the cells, or re-ligation of the linear plasmid occurring in the yeast.

We used PCR across the recombination junction to verify that homologous recombination did occur between pRRP1S and the P1. Primers were designed to pRRP1S vector sequences outside of the recombinogenic arms. These were paired with primers designed to P1 sequences located between the recombinogenic ends and the BamHI cloning site (Fig. 1A). Primer pairs 1 and 2 amplify a band of 782 bp from yeast DNA isolated from Cdx-2 or Hoxa-11 PCR positive colonies (Fig. 1B). This is the size expected for the junction between the P1s and pRRP1S. Similarly, primer pairs 3 and 4 gave the expected 778 bp band. Thus, we have rescued the P1 insert into pRRP1S by precise homologous recombination.

Total genomic DNA was isolated in agarose plugs from PCR positive yeast cells (9). The plugs were digested with AgarACE (Promega), dialyzed against TE and 5 µl were used to electotransform Escherichia coli strain DH10B (4). Transformants were selected for chloramphenicol resistance (12.5 µg/ml). Plasmid DNA was extracted from the bacteria by a standard alkaline lysis procedure (10). Plasmid DNA from bacteria and total genomic DNA from yeast was restriction enzyme digested, Southern blotted and probed for either the Cdx-2 (Fig. 2A) or Hoxa-11 genes (Fig. 2B). Using two different restriction enzymes, recombinant plasmids isolated from both yeast and bacteria produce identical sized bands as the original P1 clone (Fig. 2A and B). In addition, the ethidium bromide stained gel of restriction enzyme digests comparing the original P1s to the recombinant plasmids indicates that the banding patterns are almost identical (data not shown). The only differences in banding pattern are due to the different vectors used. Thus, the inserts in the recombinants are not rearranged.

Figure 2. Southern analysis comparing original P1 clones to recombinants in bacteria and yeast. DNAs were digested with either BamHI (odd lanes) or EcoRI (even lanes) and run on a 1% agarose gel. Lanes 1 and 2, total yeast genomic DNA from the yeast recombinants; lanes 3 and 4, the original P1 clone; lanes 5 and 6, plasmid DNA of the recombinant isolated from bacteria. Kb, position and size of 1 kb ladder molecular weight marker (Gibco) bands are indicated. (A) Recombinants from co-transformation of P1-cdx2 and pRRP1S. The blot was hybridized with Cdx-2 probe. Lanes 1 and 2 were exposed to X-ray film for 1 week. Lanes 3-6 were exposed for 18 h. (B) Recombinants from co-transformation of P1-ha11 and pRRP1S. The blot was hybridized with Hoxa-11 probe and exposed to X-ray film for 24 h.

The recombinant plasmids isolated from bacteria were also digested with I-SceI, a meganuclease with an 18 bp recognition sequence located at either end of the polylinker of pRRP1S (Fig. 1). Digestion with this enzyme should release the entire insert from the vector. Using Field Inversion Gel Electrophoresis (FIGE) and Southern blotting, the sizes of the Cdx-2 and Hoxa-11 inserts in pRRP1S were found to be 78 and 70 kb, respectively (Fig. 3). These insert sizes cannot be directly compared to those of the original P1 since there is no enzyme site available in pAd10-SacBII to release the intact inserts. However, by adding up the fragments generated by restriction enzyme digest of the P1 clones, we have estimated that the inserts are approximately equal to the sizes in pRRP1S. Thus we have successfully transferred intact inserts of at least 78 kb from the P1 clones into pRRP1S.

Figure 3. Southern analysis of FIGE to determine size of inserts in pRRP1S. I-SceI digested recombinant plasmid was run in FIGE at 200 V for 18 h at 10°C with a 1-6 s pulse time and a 3:1 forward/reverse ratio using a Switchback Pulse Controller (Hoefer Scientific). Southern blotted DNA was probed with either the Cdx-2 (lane 1) or Hoxa-11 (lanes 2 and 3) probe and exposed to X-ray film for 18 h. Lane 1, plasmid DNA isolated from bacteria from recombination between P1-cdx2 and pRRP1S; lanes 2 and 3, plasmids isolated from two separate bacteria clones from recombination between P1-ha11 and pRRP1S. Kb, position and size of Midrange PFG Marker I (New England Biolabs) bands are indicated.

We have shown that the insert of the P1 vector, pAd10-SacBII, can be transferred to the yeast-bacteria shuttle vector, pClasper, by co-transformation mediated homologous recombination in yeast. We can now insert a reporter gene or mutate the genes in pClasper by homologous recombination in yeast as previously shown (5). The sequence of the recombinogenic ends in the targeting vector, pRRP1S, are also homologous to the region surrounding the linker of the PAC vector, pCYPAC-1 (2). Thus, this vector can also be used to clone inserts from PACs. Since the recombinogenic ends in pClasper are made by PCR, it will only require amplification and cloning of homologous regions surrounding the inserts in BACs to apply the same method to transfer inserts from the BAC vector into pClasper.

ACKNOWLEDGEMENTS

We thank C. S. Shashikant and Frank Ruddle for the gift of the Cdx-2 P1 clone and Ansel Zhao and Steven Potter in collaboration with Jeffrey Innes for the gift of the Hoxa-11 P1 clone.

REFERENCES

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*To whom correspondence should be addressed. Tel: +1 513 556 9723; Fax: +1 513 556 5299; Email: suzanne.bradshaw@uc.edu

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