Fig. 3.

Targeted and heritable addition of the SP110 gene using TALE nickase. (A) Schematic representation of the gene-targeting vector. (B) Schematic overview depicting the targeting strategy for SP110. D450A, FokI bearing a D450A mutation. (C) Schematic overview screening the individual colonies. 5j F, lr F, and 3j R, lr R are primers for regions outside the homologous arms; 5j R and 3j F are primers for the targeting vector region. Southern blot probes are shown as red lines; Hind III digestion is used in Southern blot analysis. (D and E) Southern blot analysis of the nine heterozygous donor cells used for SCNT. (D) A 6.8-kb band resulting from targeted inclusion of the SP110 cassette was detected in addition to the 5.9-kb wild-type band when probe 1 was used. (E) Only a 6.8-kb targeted band was detected when probe 2 was used.