Southern blot analysis of three different GATA-2 BAC clones modified by the G2TC DNA fragment. Newly introduced restriction sites (Fig. 1D) were used to digest both wild-type (lanes 1) and modified (lanes 2) BAC DNA. (A) GATA-2 BAC clone 3 was digested with the indicated enzymes and probed with a 2-kb wild-type ClaI-EcoRV genomic fragment (Fig. 1B). A 9-kb wild-type EcoRV fragment became 8.8-kb and 2-kb fragments. An 11-kb wild-type XhoI-BamHI fragment became 9-kb and 2-kb fragments. A 20-kb wild-type SalI fragment became 12-kb and 8.6-kb fragments. A 10.5-kb wild-type ClaI fragment became 9.5-kb and 1.7-kb fragments. A 13.5-kb wild-type HindIII fragment became 12-kb and 2.5-kb fragments. GATA-2 BAC clones 1 and 4 (B) also contained the desired gene replacement.

GFP expression from modified GATA-2 BACs in living zebrafish embryos. (A) GFP expression driven by modified BAC clone 4 in the ventral ectoderm and mesoderm of a 6-h shield stage embryo. Arrow indicates the dorsal shield. (B) GFP expression driven by modified BAC clone 4 in the posterior intermediate cell mass of a 20-h embryo. (C) GATA-2 expression pattern in an 18-h embryo as detected by RNA in situ hybridization. (D) GFP expression driven by modified BAC clone 3 in circulating hematopoietic cells of a 48-h embryo. GFP expression driven by modified BAC clone 4 in the EVL (E and G) and in a motoneuron (F) of 48-h embryos.