ROSA26 locus–targeted Flt-1 isoform rescue clones. (A) Flt-1 isoform transgene ROSA26 targeting. (top) Restriction map of the ROSA26 locus. (middle) pROSA26-1–containing insertion cassette of PECAM promoter/enhancer, Flt-1 isoform, and a hygromycin B selectable marker with PGK–diptheria toxin A cassette for negative selection. (bottom) Predicted structure of the ROSA26 locus after integration of the pROSA26-1 insertion cassette in flt-1^−/− ES cells. Arrows denote primers used to verify correct targeting into flt-1^−/− ES cells (not depicted). (B) Diagram of individual Flt-1 isoforms. Soluble Flt-1 (sFlt-1) and membrane-localized Flt-1 (mFlt-1) each contain an extracellular VEGF-A–binding domain consisting of Ig domains (gray circles). Only mFlt-1 contains a transmembrane domain region (TM) and a tyrosine kinase domain. (C) Total RNA was isolated from differentiated day 8 wild-type (WT), flt-1^−/−, flt-1^{−/−;Tg ROSA-PECAM-sflt-1}, and flt-1^{−/−;Tg ROSA-PECAM-mflt-1} cultures and analyzed by semiquantitative RT-PCR using flt-1 primers.