V K Singh1, G J Chader, I R Rodriguez. 1. Laboratory of Retinal Cell and Molecular Biology, National Eye Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.
Abstract
PURPOSE: Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mouse PEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mouse PEDF cDNA was cloned from a mouse liver cDNA library using human PEDF cDNA as a probe. The mouse PEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mouse PEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mouse PEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and human PEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.
PURPOSE:Pigment epithelium-derived factor (PEDF), a neurotrophic protein, is a member of the serine protease inhibitor supergene family. PEDF promotes both neuronal phenotype in cultured Y79 cells and neuronal survival in cultured cerebellar granulocytes. The purpose of this study was to clone the mousePEDF gene and to determine its structure and levels of expression in different tissues. METHODS: The mousePEDF cDNA was cloned from a mouse liver cDNA library using humanPEDF cDNA as a probe. The mousePEDF gene was cloned from a mouse ES genomic P1 library. DNA sequencing was performed using a PE-Applied Biosystems model 373 automated fluorescent sequencer. RESULTS: The mousePEDF cDNA is 1461 bp in length and contains an open reading frame of 417 amino acids. The mousePEDF gene spans approximately 13 kb and, like the human, it is fragmented into 8 exons. The splice sites follow the AG/GT consensus rule. Southern blot analysis indicates that the mouse genome contains only one gene for PEDF. Northern blot analysis shows the presence of the PEDF transcript in a broad range of adult mouse tissues with liver showing the highest level of expression. CONCLUSIONS: The mouse and humanPEDF promoters share overall 27% similarity but are nearly identical between mouse +86 to -166 and human +102 to -96. The present study will allow us to move from in vitro experiments to in vivo studies through the development of a "knock-out" mouse model.
Authors: Virginia Haurigot; Pilar Villacampa; Albert Ribera; Assumpcio Bosch; David Ramos; Jesus Ruberte; Fatima Bosch Journal: PLoS One Date: 2012-07-20 Impact factor: 3.240