PTEN (or MMAC1/TEP1) tumor suppressor gene is frequently mutated in a variety of human cancers and in three cancer predisposition syndromes (Eng and Peacocke, 1998; Dahia, 2000). PTEN negatively regulates the phosphatidylinositol 3-kinase (PI3 kinase) signaling pathway by dephosphorylating PIP3, the product of PI3 kinase (for review, see Cantley and Neel, 1999). Inactivation of Pten (chromosome 19) in mouse models confirmed PTEN to be a bona fide tumor suppressor (Di Cristofano et al., 1998; Podsypanina et al., 1998; Suzuki et al., 1998; Lesche et al., submitted): Pten+/J mice developed tumors in multiple organs and PtenJ/J mice died during embryogenesis before midgestation. To overcome the early lethal phenotype in PtenJ/J mice and to study the roles of PTEN in embryonic development, adult tissue function, and tumorigenesis, we have generated a conditional Pten knockout mouse strain. LoxP sequences were inserted into the endogenous Pten locus flanking exon 5 as illustrated in Figure 1. Exon 5 encodes the phosphatase domain of PTEN in which many tumor-associated mutations have been detected. PtenloxP/+ ES cells were injected into either C57/B6 or Balb/c blastocysts. Chimeric mice were backcrossed to either C57/B6 or Balb/c mice and germ-line transmission of the PtenloxP/+ allele was confirmed by Southern blot and PCR genotyping (not shown). In contrast to the embryonic lethal phenotype observed in PtenJ/J mice, PtenloxP/loxP animals were viable. Normal PTEN level and function were detected in PtenloxP/loxP MEF cells and no spontaneous tumor formations were observed up to two years, suggesting that introducing the loxP sites into the Pten locus does not perturb the normal function of PTEN. To demonstrate Cre-mediated exon 5 deletion, we crossed PtenloxP/loxP animals with the GFAP-Cre transgenic mice (Zhuo et al., 2001) aimed for brain-specific deletion. As shown in Figure 2c, Cre expression in the PtenloxP/+; GFAP-Cre+/J mice resulted in neural-specific excision of exon 5 (lanes 1–5). In contrast, very low or no excision could be detected in other nonneural tissues (lanes 6–9). Finally, we showed that no PTEN protein could be detected in conditional deleted tissue and the known downstream signaling molecule AKT/PKB was hyperphosphorylated (Fig. 2c). Thus, the PtenloxP/loxP mouse line generated will be valuable for studying the function of PTEN in animal development and tumorigenesis.