Design and construction of the targeting vectors. (A) Diagram shows the 2-step approach for developing a murine model system to study targeted gene correction. The first step involved the generation of mouse ES cells containing the β^6I mutant globin gene. The structure of the wild-type β-globin gene locus is shown on the top line (β^maj genomic allele), and the targeting vector that was used to introduce the β^6I mutation is shown on the second line (β^6I targeting vector). The structure of the targeted allele with a retained PGK-neo is depicted on the third line (β^6I + PGK-neo allele), and shown below is the structure of the allele after PGK-neo is excised by Cre recombinase (β^6IΔPGK-neo allele). In step 2, the ES cells heterozygous for the β^6IΔPGK-neo mutant allele were used to determine whether correcting vectors of different sizes could correct the β^6I mutation via homologous recombination. The corrected β-globin locus is depicted on the bottom line (corrected β^maj allele). The location of the internal probe is shown (black bar). After EcoRV and BglII double digestion of G418-resistant ES cell DNA, the β^6IΔPGK-neo mutant allele yields a 2.7-kb fragment, and the wild-type/corrected allele produces a 3.4-kb fragment. The disappearance of the diagnostic 2.7-kb band indicates that targeted correction of the mutant allele has occurred. Insertion of the correcting vector DNA into a random site(s) of the genome is expected to give rise either to fragments of random sizes, or occasionally, to produce a more prominent 3.4-kb wild-type band without the loss of the 2.7-kb mutant band. B indicates BglII; E, EcoRV. (B) Vector construction. pβC110:A 1.5-kb AvaI fragment containing phosphoglycerate kinase 1 gene promoter-driven neomycin resistance gene (PGK-neo) was inserted into an AvaI site 4.1 kb downstream from the cap site of β-major gene in P1 1935. PGK-neo was inserted in the same transcriptional orientation as that of the globin genes. pβC24: A 24-kb PvuII fragment containing the entire β-major gene was isolated from the P1 1935 DNAand subcloned into the SmaI site of pGEM7zf(–) (Promega, Madison, WI). The identical AvaI PGK-neo–containing fragment was then inserted at the same AvaI site as described. pβC16: A 7.9-kb fragment, including the 7.8 kb in the 5′ portion of the 24-kb β-major gene insert and a small region containing the multicloning sites, was removed from pβC24 by HindIII partial digestion (loss of 3 contiguous 0.3-, 6.4-, and 1.2-kb HindIII fragments). Circularization of the remaining portion of the vector gave rise to the pβC16. pβC8: A 6-kb AvaI fragment spanning the mouse β-major gene was isolated from P1 1935 and subcloned into an AvaI site that is located upstream from a PGK-neo in pCR II (Invitrogen, Carlsbad, CA). Using P1 1935 as the template, a 2.2-kb fragment, located between 4.1 kb and 6.3 kb downstream from β-major cap site, was generated by polymerase chain reaction (PCR). Flanking BamHI-HindIII sites were introduced to facilitate the insertion of this fragment into BamHI-HindIII sites downstream from PGK-neo. The left arm, PGK-neo cassette, and right arm were cloned in the proper orientation. No β-globin locus DNA sequences were removed from pβC8. All the targeting vectors were linearized before use (pβC110 with SalI; pβC24 and pβC16 with XhoI; pβC8 with ApaI). The map of the mouse β^6IΔPGK-neo mutant allele is shown on the top line (β^6I allele), and the structure of the targeting vectors containing 110 kb (pβC110), 24 kb (pβC24), 16 kb (pβC16), and 8 kb (pβC8) of targeting DNAare depicted below. (C) Structural analysis of the mouse β-globin gene locus. The P1/PAC clone 1935 (top line; P1 1935) and the PGK-neo-containing β-globin locus within the targeting vector pβC110 (bottom line; pβC110) are shown. The sizes of relevant restriction fragments (in kb) are shown below the diagrams of β-globin loci. The pβC110 vector was generated by insertion of a 1.5-kb AvaI fragment containing a PGK-neo cassette (hatched box) into an AvaI site (designated as A*) downstream from the β-major globin gene within the P1 1935. Insertion of PGK-neo led to an increase in the size of some restriction fragments, including a BglII fragment from 2.8 kb to 4.3 kb, an EcoRV fragment from 3.7 kb to 5.2 kb, and an HpaI fragment from 3.0 kb to 4.5 kb. Addition of PGK-neo, which contained an internal NcoI and a PstI site, disrupted an 8.1 kb NcoI and a 4.3 kb PstI fragment, and produced 2 diagnostic 4.8-kb NcoI fragments, and 2 PstI fragments of 3.6 kb and 2.3 kb in length. B indicates BglII; E, EcoRV; H, HpaI; N, NcoI; P, PstI; A, AvaI). (D) Restriction analysis of P1 1935 and pβC110. Plasmid DNA was digested with BglII, EcoRV, HpaI, NcoI, and PstI, and the resultant restriction fragments were analyzed by agarose gel electrophoresis. The data revealed that the pβC110 vector contained a single PGK-neo cassette that had been properly inserted as predicted in panel C. The correct orientation of PGK-neo in the locus was revealed by DNA sequence analysis of the insert-locus junctions with sequencing primers located both within the locus and within the PGK-neo. Similar structural and sequence analyses were also performed on other targeting vectors to verify the correct vector configuration (data not shown).