Identification of a novel proliferation-inducing determinant using lentiviral expression cloning.
One of the major challenges in the post-genome era is the correlation between genes and function or phenotype. We have pioneered a strategy for screening of cDNA libraries, which is based on sequential combination of lentiviral and oncoretroviral expression systems and can be used to identify proliferation-modulating genes. Screening of a lentiviral expression library derived from adult human brain cDNA resulted in cloning of the potent proliferation-inducing determinant termed pi1 (proliferation inducer 1). Transduction experiments using GFP-expressing oncoretroviruses to target proliferation-competent cells suggested that overexpression of pi1 initiates proliferation of human umbilical vein endothelial cells (HUVECs). Growth induction of HUVECs as well as Swiss3T3 fibroblasts was confirmed by Brd-uridine incorporation assays, which correlated increased DNA synthesis with expression of pi1. The identified pi1 cDNA is 297 bp long and encodes a 10 kDa polypeptide. Since deregulation of proliferation control accounts for a number of today's untreatable human diseases such as neurodegenerative disorders and cancer, discovery of novel proliferation-modulating genes is essential for developing new strategies for gene therapy and tissue engineering.
Chilov
Dmitri
D
Institute of Biotechnology, Swiss Federal Institute of Technology Zurich, ETH Hoenggerberg, HPT D74, CH-8093 Zurich, Switzerland.
Fux
Cornelia
C
Joch
Hana
H
Fussenegger
Martin
M
D016475:3T3 Cells; D000328:Adult; D000818:Animals; D001921:Brain / Q000378:metabolism; D002455:Cell Division / Q000235:genetics; D002460:Cell Line; D002478:Cells, Cultured; D003001:Cloning, Molecular / Q000379:methods*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics*; D005434:Flow Cytometry; D015870:Gene Expression; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D016086:Lentivirus / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D014162:Transfection
D018076:DNA, Complementary; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins