Quantification of HGF levels in Tie2 KO embryos. (A) Real time RT-PCR analysis on HGF levels was performed on wild-type and Tie2 knockout embryo yolk sacs harvested at day 9.5. Gene expression levels were normalized according to the expression of the housekeeping gene GAPDH. HGF expression was decreased 4.26-fold in Tie2-null yolk sacs compared with wild-type controls. Three pairs of embryo tissues were used in the study. RT-PCR was performed in triplicate and with 3 different dilutions of cDNA. *P < .01 compared with control. (B) RT-PCR analysis on HGF was also performed on endothelial cells isolated from the wild-type yolk sac (C166). Two sets of primers for HGF were used to increase specificity. HGF expression was confirmed (lane 2, 300 bp; lane 3, 500 bp). GAPDH was used as a control (lane 1, 440 bp).