Figure 3. Gene targeting of the endogenous human beta-globin locus.

A. Schematic overview of gene targeting strategy for the human beta-globin locus. The sickle cell mutation is labeled by an asterisk and ZFN target sites are indicated by arrows. The desired recombination event inserts a PGK promoter-puromycin-resistance cassette or PGK promoter-neomycin-resistance cassette flanked by loxP sites (black triangles) into the intron between exons 1 and 2. Southern blot probes are indicated by grey bars and PCR primers are indicated by arrows. Not drawn to scale.

B. Representative PCR analysis of puromycin-resistant clones. PCR of gene targeted clones gives a band of 1.4 kb when amplified with 3′ primers and 1.6 kb with 5′ primers. Non-targeted clones fail to give PCR product.

C. Southern blot using a 5′ external probe on genomic DNA digested with PvuII. A beta-globin allele that has not undergone gene targeting gives a 12.8kb band while a targeted allele gives a 7.8kb band due to the presence of a PvuII site in the puromycin-resistance gene. Note that after expansion in culture, four clones previously revealed to be mixed populations by sequencing no longer possess the gene targeted allele.

D. Sequence of gene targeted allele detected by PCR assay. The allele that has undergone homologous recombination with the donor template is wild-type at the E6 codon that is mutated in sickle cell disease, contains translationally silent mutations incorporated into ZFN target site 1, and has incorporated the puromycin or neomycin-resistance cassette.

E. Sequence of the non-gene targeted allele. The untargeted allele in clones IA12, IA13, IA38, IB20, IB48, IB49, IB50, IC54 and IIBI is the allele that does not harbor the sickle cell mutation, indicating that the sickle cell allele underwent gene targeting. In clone IA5, the untargeted allele is the sickle cell allele. It was not possible to determine which allele underwent gene targeting in clones IA54, IB54, IC43 or IC52 presumably because these were mixed populations of both targeted and untargeted clones.

F. Table showing the number of puromycin or neomycin-resistant colonies picked for each experiment and the percentage of these that were determined to be correctly targeted by the PCR screening assay. Asterisk indicates that ZFNs were expressed as fusions to the heterodimeric ELD/KKR FokI nuclease domain as opposed to the heterodimeric EL/KK FokI nuclease domain (no asterisk).