Establishment of pten knockout medaka with transcription activator–like effector nucleases (TALENs) as a model of PTEN deficiency disease

Abstract

Phosphatase and tensin homolog (PTEN) is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K)–AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator–like effector nuclease (TALEN) technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko) embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf) and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT) in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

Phosphatase and Tensin Homolog (PTEN) of Japanese Flounder—Its Regulation by miRNA and Role in Autophagy, Apoptosis and Pathogen Infection

“…Another miRNA, miR-21, could decrease autophagy by down-regulating PTEN in mesangial cells [ 37 ]. In fish, it has been reported that zebrafish PTEN deficiency induced the activation of PI3K/mTOR pathway and AKT, which affected embryo and tissue development [ 38 , 41 ]; madaka PTEN knockout also upregulated PI3K/AKT signaling, which led to abnormal embryo development [ 42 ]. In the present study, we found that compared with human and mouse PTEN, fish PTEN homologs are highly conserved in sequence and structure, and, in flounder, PoPTEN was constitutively expressed in a wide range of tissues.…”

“…In fish, studies on PTEN have been documented mainly in zebrafish, in which it was shown that PTEN played an important role in embryonic development, caudal fin-fold regeneration, tumorigenesis, hematopoiesis, and myelocyte migration [ 38 , 39 , 40 , 41 ]. In medaka, PTEN was also shown to be involved in embryo development [ 42 ].…”