THE RAY- MANUAL. Instructions for the construction of complex targeting vectors using RAY (rapid assembly in yeast) Thorsten Storck December '96

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1 Thorsten Storck December '96 THE RAY- MANUAL Instructions for the construction of complex targeting vectors using RAY (rapid assembly in yeast) Principle of the method Genetic elements (selection markers, transcriptional control sequences, indicator genes, etc.) and mutations are introduced into a gene segment cloned into a yeast shuttle vector by homologous recombination in yeast (Storck et al. 1996). By relying on recombination this method does not require suitable restriction sites and overcomes the need for extensive restriction mapping of the gene segment. The recombination event is triggered and directed by bp sequences (recombinogenic arms), which have to be amplified by PCR from the gene segment and cloned into an appropriate plasmid in order to flank the donor DNA containing the genetic elements. The donor DNA is released from the plasmid and cotransformed into yeast together with the shuttle vector harbouring the gene segment. Recombination between the recombinogenic arms and their homologous sequences on the gene segment introduces the genetic elements into the desired position. For the design of the recombinogenic arms you should note that i) any basepairs, that seperate the sequences from which the recombinogenic arms are derived, will get lost from the gene segment during the recombination event, allowing for precisely designed deletions. ii) mutations engineered into the recombinogenic arms will get incorporated into the gene segment with a frequency inversly related to their distance from the genetic elements. Selection strategies The recombination frequencies we observed are high enough to allow a PCR-based identification of positive yeast colonies without prior selection (routinely 1-2% of transformed yeast cells contain the recombination product). One primer specific for a genetic element and one binding next to the recombinogenic arm sequence on the gene segment are needed for PCR, which may be performed using patches of yeast colonies, precluding the need for DNA preparations. However, about yeast colonies have to be streaked onto a grid plate and screened by PCR in order to isolate several independent recombination products. Less laborious is our favourite strategy, which offers a tight selection in yeast by introducing the yeast selection marker URA3 together with the genetic elements. After the cotransformation, yeast cells are plated on Ura-dropout plates. More than 95% of yeast colonies growing on these plates contain the recombination product. To apply this strategy, you should use the plasmid pray-1 for the assembly of the donor DNA. This plasmid offers a dual-selection box, containing the URA3 marker and the neomycin resistance marker for selection in embryonic stem cells. The dual-selection box is flanked by loxp-sites and suitable restriction sites for the introduction of additional genetic elements and recombinogenic arms. A third strategy is based on a neomycin resistance gene, modified to confer neo resistence to

2 E. coli as well as ES-cells, permitting a selection for the recombination product in E.coli. Cotransformed yeast colonies are pooled, extrachromosomal DNA is prepared and electroporated into E. coli. Bacterial transformants containing the recombination product are selected on plates containing kanamycin. To apply this principle, you can use the plasmid pray-2 which is identical to pray-1, with the exception that the dual-selection box has been replaced by the modified neo gene. prerequisites In order to construct targeting vectors with RAY you need i) a phage or plasmid clone containing a segment of your target gene, long enough to provide the homologous regions needed for recombination in ES-cells. This gene segment will contain the region, into which the genetic elements are to be introduced. ii) sequence information of at least 250 bp up- and downstream of the site for the insertion of the donor-dna how to get started shuttle vector Subclone a suitable segment of your gene of interest into a yeast shuttle vector (e.g. YCplac22). Take into account that, after the introduction of the genetic elements, you will need a unique restriction site to linearize the targeting vector prior to ES-cell electroporation. donor DNA Introduce appropriate genetic elements and recombinogenic arms into the restriction sites flanking the selection box of pray-1 or pray-2. Release the donor DNA by a restriction digest, for example with SfiI/NotI, and purify the DNA fragment from an agarose gel. Important note: The BstEII site in the polylinker of the RAY-vectors shares homology with the consensus sequence for splice donor sites! Take suitable precautions! transformation of yeast We use the yeast strain DF5, which posesses mating type a/alpha and the genotype his3-200, leu2-112, lys2-801, trp1-1(am), ura3-52 his3-200, leu2-112, lys2-801, trp1-1(am), ura3-52 but we guess that other laboratory strains will perform equally well if they allow for Trp and Ura selection. Dilute a yeast overnight culture in 50ml YPD-medium to an OD600 of 0.2 and incubate in a shaker at 30 C until it reaches an OD600 of Transfer culture into a 50ml Falcon tube and harvest the cells by centrifugation (2500rpm in a Heraeus-Christ centrifuge, 10min, RT). Resuspend pellet in 10ml water and centrifuge as above. Resuspend pellet in 10ml LiAc-solution (10mM Tris-Cl ph8, 1mM EDTA, 100mM Li-

3 acetate) and centrifuge as above. Resuspend pellet in 250µl LiAc-solution. You have now enough competent yeast cells for seven transformations. The cells should be used immediately but may be kept on ice for a few hours. For the cotransformation mix 50µl competent yeast cells 2µl salmon sperm DNA (high molecular weight, denatured) 500ng shuttle vector harbouring the gene segment 500ng donor DNA 300µl PEG-solution (10mM Tris-Cl ph8, 1mM EDTA, 100mM Li-acetate,40% PEG) and agitate in a rototorque for 30min at 30 C. Perform a heat shock for exactly 15min in a 42 C water bath with shaking every 3min. Plate 100µl of the transformation mix onto either a Trp dropout plate, if you don t select for the recombination product in yeast, or onto a Trp/Ura dropout plate, if you take advantage of the URA3 selection marker. Incubate at 30 C for 2-3 days. Identification of positive yeast colonies by PCR This applies for the "no selection strategy" as well as for yeast transformants selected for the Ura + -phenotype, with the notable difference, that in the former case colonies have to be screened, whereas in the latter, you may expect that nearly every Ura + -colony contains the recombination product. For convenience, yeast colonies are streaked onto grid plates and incubated at 30 C for 2 days. Transfer patches of these colonies from the grid plate into PCR tubes containing 10µl water. Incubate for 10min at 96 C. Add the following components 2µl 10x PCR buffer (200mM Tris-HCl ph 8.4, 500mM KCl) 1µl dntps (5mM each) 1µl 50mM MgCl2 0.5µl primer A (specific for a genetic element) 0.5µl primer B (specific for a sequence next to a recombinogenic arm on the gene segment) 5µl water 0.25µl taq polymerase (5U/µl) and run the PCR with the following conditions: denaturation: 94 C, 5 min 40 cycles: 94 C, 45 sec 60 C, 1min (primer dependent)

4 end polish: 72 C, 1min 72 C, 10 min For scientific accuracy, both recombination sites should be checked with specific primer pairs, but we never found an example, where the recombination machinery had involved only one recombinogenic arm. Transfer of the targeting vector to E.coli Start small scale cultures in 3-5 ml of selection medium (Trp dropout or Ura/Trp dropout) from positive yeast colonies. After 2 days in a 30 C shaker, transfer 1.5 ml into an Eppendorf tube and harvest the cells by centrifugation (13000g, 10 sec). Decant supernatant and redissolve the pellet in the remaining liquid. Add 0.2 ml Yeast-Lysis-Buffer (10mM Tris-Cl ph8, 1mM EDTA, 100mM NaCl, 1% SDS, 2% TritonX100) 0.2 ml phenol/chloroform/isoamyl alcohol (25:24:1) glassbeads (500µm, acid-washed, Sigma) up to the interphase and vortex hard for 2 min. Spin at 13000g for 5 min. Precipitate 100µl of the supernatant with Na-acetate and EtOH, wash and resuspend in 20µl TE. Electroporate 2µl into competent E. coli (we use strain DH5alpha) and plate onto ampicillin containing plates. Not all colonies growing on these plates will contain the targeting vector, since a positive yeast colony harbours in addition to the recombination product one or several copies of the non-recombined shuttle vector. In our hands 30-90% of bacterial transfomants contained the targeting vector, which can be discriminated from the shuttle vector via restriction digests. Finally, start a large-scale culture and prepare high-quality DNA for ES-cell electroporation. The use of pray-2 for selection in E. coli Following the yeast transformation let the colonies grow on Trp dropout plates for 4-5 days at 30 C. Harvest colonies by rinsing the plate with 1.8ml water and transfer the suspension to a 2ml Eppendorf tube. Isolate the extrachromosomal DNA using the protocol described above but scaled up two-fold. After the EtOH-precipitation resuspend the pellet in 20µl water. Electroporate 2µl into competent E. coli (efficiency >5x10 9 /µg, we use XL2-Blue, Stratagene). Allow cells to recover for 3 hrs at 37 C in 1ml SOC-medium with gentle agitation. Spin down and resuspend pellet in 200µl SOC-medium. Plate 1/10th on a LB-plate containing 200mg/ml ampicillin to determine the transformation efficiency. More than 250 colonies should come up. Plate the remainder on LB-plates containing 5µg/ml kanamycin.

5 Start small scale cultures in TB-medium containing ampicillin from the kanamycin resistent colonies and check your targeting vector by restriction mapping. Media For yeast media and plates see for example: Ausubel, F.M., Brent, R., Kingston, R.E., Moore, D.D., Seidman, J.G., Smith, J.A. and Struhe, K. (eds.) Current Protocols in Molecular Biology. John Wiley and Sons, Inc., Massachusetts, Vol. II, Chapter 13. Composition of media and plates for E. coli is found for example in: Sambrook, J., Fritsch, E.F. and Maniatis,T.(1989) Molecular Cloning: A Laboratory Manual, Cold Spring Harbour Laboratory Press References Storck, T., Krüth, U., Kolhekar, R., Sprengel, R. and Seeburg, P.H., Rapid construction in yeast of complex targeting vectors for gene manipulation in the mouse, Nucleic Acids Research 24, (1996) The nucleotide sequences of pray-1 and pray-2 are available from the GenBank (accession numbers U63018 and U63120). If you have any further questions feel free to contact me at: Storck@sun0.urz.uniheidelberg.de Good luck!