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. Author manuscript; available in PMC: 2011 Sep 1.

Published in final edited form as: Mech Ageing Dev. 2010 Aug 12;131(9):562–573. doi: 10.1016/j.mad.2010.08.001

Fig. 4 — Plasmid-chromosome targeted recombination assay. (A) Diagram of pAG6ins0.9 targeting vector. This vector contains a full-length copy of the CHO APRT gene (dark grey bar) interrupted by 945bp of heterologous sequence (black bar) at the XhoI site located in exon 3. The plasmid was linearized by digestion with SalI to generate an ends-in targeting configuration where 271nt and 670nt flank the double-strand break. The vector backbone contains a GPT allele used to select for non-targeted recombinatiton events. The target APRT locus (light grey) in ATS-49tg has an inactivating 3bp deletion mutation in exon 5, which destroys a MboII restriction site. Relevant restriction sites are shown: B-BamHI; Bg-BglII; RV-EcoRV; H-HindIII; Mb-MboII; S-SalI; X-XhoI. (B) Plating cells into ALASA media selects for APRT⁺ rcombinants. Three possible classes of recombinants are diagramed in addition to an aberrant class.

Fig. 4 — Plasmid-chromosome targeted recombination assay. (A) Diagram of pAG6ins0.9 targeting vector. This vector contains a full-length copy of the CHO APRT gene (dark grey bar) interrupted by 945bp of heterologous sequence (black bar) at the XhoI site located in exon 3. The plasmid was linearized by digestion with SalI to generate an ends-in targeting configuration where 271nt and 670nt flank the double-strand break. The vector backbone contains a GPT allele used to select for non-targeted recombinatiton events. The target APRT locus (light grey) in ATS-49tg has an inactivating 3bp deletion mutation in exon 5, which destroys a MboII restriction site. Relevant restriction sites are shown: B-BamHI; Bg-BglII; RV-EcoRV; H-HindIII; Mb-MboII; S-SalI; X-XhoI. (B) Plating cells into ALASA media selects for APRT⁺ rcombinants. Three possible classes of recombinants are diagramed in addition to an aberrant class.