Generation of Floxed Tissue Factor Mice.

Tissue factor (TF) is the primary cellular initiator of blood coagulation. Since TF Deficiency leads to embryonic lethality, we generated floxed TF mice to allow analysis of the effect of deleting the TF gene in different cell types. A targeting vector was constructed containing two loxP sites spanning the promoter and intron 1 together with a neomycin resistance gene (NEO) in intron 1 that is flanked by two frt sites. The floxed TF gene targeting plasmid was transfected into ES cells and clones were selected using G418 and gancyclovir. We screened 144 clones by Southern blotting and found six targeted clones. Three clones were injected into blastocysts to generate chimeric mice. Only one chimera demonstrated germline transmission when bred with C57BL/6 mice. TF^flox/flox mice were generated at the expected frequency (15 out of 58; 25.8%) from TF^+/flox intercrosses, indicating that the floxed TF allele is functional. To detemine if the DNA between the loxP site can be efficiently excised from the floxed TF allele to create an inactive gene, we crossed TF^flox/flox mice with mice expressing the Cre transgene under the control of the adenovirus EIIa promoter. This transgene is expressed in one cell zygotes and results in deletion of DNA at the earliest stage of development. From this breeding, we generated TF^+/flox− offspring and intercrossed them. To date, analysis of 14 mice at wean has not identified a TF^{flox−/flox−} mouse, suggesting efficient inactivation of the TF gene. To remove the NEO cassette from intron 1 of the floxed TF allele, we crossed TF^flox/flox with transgenic mice expressing flipase under the control of the human β-actin promoter (FLP). TF^+/flox/FLP⁺ mice were intercrossed. To date, we have successfully generated 9 TF^flox/flox/NEO⁻ mice. These mice will be crossed with tissue-specific deleter strains to investigate the contribution of TF expression in various cell types to a variety of biological processes, including hemostasis, inflammation, angiogenesis, metastasis and cell migration.