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KMID : 0390320120220010125
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Chungbuk Medical Journal
2012 Volume.22 No. 1 p.125 ~ p.130
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Preparation of a Lentivirus expressing Salvador homolog 1
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Park Byeong-Hee
Lee Yong-Hee
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Abstract
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Purpose: Mammalian ste20-like kinase (MST) signaling pathway including Salvador homolog 1 (SAV1) is an important dual regulator of apoptosis and cell proliferation and is emerging as a novel tumor suppressor pathway. The aim of this study was to prepare a lentivirus harboring SAV1 gene in order to transduce SAV1 gene into the cells that are resistant to transfection of foreign genes.
Materials and Methods: SAV1 cDNA with 3 myc tag was subcloned into the lentiviral transfer vector, pFUGW which includes a GFP expression cassette. HEK293 cells were transfected with this plasmid to confirm SAV1 expression. Lentivirus was prepared by transfection of 293T cells with pFUGW-3XMyc-SAV1, envelope glycoprotein expression vector, and packaging vector.
Results: After transfection of HEK293 cells with pFUGW-3XMyc-SAV1, SAV1 expression was confirmed by GFP fluorescence and immunoblotting with myc antibody. HeLa and A549 cells transduced with SAV1-lentivirus showed strong GFP fluorescence. Fully differentiated 3T3-L1 adipocytes, that are difficult to transfect foreign genes into, were transduced with SAV1-lentivirus and they showed SAV1 expression analyzed by immunoblotting.
Conclusion: In this study, we generated and characterized a lentivirus harboring SAV1 and this will be a valuable experimental tool for the study of MST2 kinase signaling pathway.
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KEYWORD
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SAV1, MST, Lentivirus
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