BAC recombineering, gene targeting and RMCE strategies.

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BAC recombineering, gene targeting and RMCE strategies. BAC recombineering, gene targeting and RMCE strategies. (A) Steps for making a ROSA26 gene targeting vector by BAC recombineering. Short inner homology arms were amplified from a BAC containing the ROSA26 gene and cloned into the pLCA.71/2272 vector. In the first recombination step (1), the cassette containing a puΔtk fusion gene surrounded by tandem lox71 and lox2272 sites was introduced into the BAC. After cloning short outer homology arms into pMCS.DT-A, a second recombination step (2) enabled retrieval of the targeting vector. DT, diptheria toxin A; MCS, multiple cloning site; HA1/2, short homology arm 1 or 2. (B) Generation of the ROSA26 exchange vector by BAC recombineering. pMCS.66/2272 was used to retrieve a 5.165-kb fragment of the ROSA26 gene, thereby generating a basal exchange vector. A variant of this plasmid was used to make all FP exchange vectors as shown in Fig. 2. (C) Gene targeting and RMCE strategy. A ROSA26LCA allele containing the puΔtk fusion gene flanked by tandem lox71 and lox2272 sites was made by gene targeting in mESCs. Puromycin-resistant mESC clones were screened by Southern blot analysis. DNA was digested with NsiI and hybridized with the 3′ probe indicated in the figure. Correctly targeted clones containing the ROSA26LCA allele were used to perform RMCE to derive eight different FP-expressing alleles. PCR using primers 1–4 (red arrows) were used for the screening of clones that were resistant to puromycin and sensitive to gancyclovir. (D) Southern blot analysis. The presence of an 11 kb band indicates a correctly targeted mESC clone with the ROSA26LCA allele. (E) DNA PCR analysis. Representative PCR amplification results are shown, indicating correct exchange of the exchange cassette into the ROSA26LCA allele. Prior to RMCE, cells with the ROSA26LCA allele exhibit a 503 bp band after PCR using primers 1 and 2. Correctly exchanged clones exhibit both a 537 bp and 503 bp product using primers 1 and 2, and a 565 bp product with primers 3 and 4. Sara X. Chen et al. Dis. Model. Mech. 2011;4:537-547 © 2011. Published by The Company of Biologists Ltd