Document Detail


High-frequency transfer of cloned herpes simplex virus type 1 sequences to mammalian cells by protoplast fusion.
MedLine Citation:
PMID:  9279387     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The protoplast fusion technique of Schaffner (W. Schaffner, Proc. Natl. Acad. Sci. U.S.A. 77:2163-2167, 1980) has been adapted to introduce cloned herpes simplex virus genes into cultured mammalian cells. The technique involves digesting bacterial cell walls with lysozyme to produce protoplasts and then fusing the protoplasts to mammalian cells by treatment with polyethylene glycol. For monitoring transfer, protoplasts were labeled with the fluorescent dye fluorescein isothiocyanate before fusion. After fusion, greater than 50% of the mammalian cells were fluorescent, demonstrating that bacterial material was transferred with high frequency. Transfer of plasmid pBR325 occurred at frequencies of 1 to 2%, as measured by in situ hybridization. Fusion transfer of a chimeric plasmid consisting of the herpes simplex virus type 1 (strain KOS) EcoRI fragment F in pBR325 resulted in expression of some viral genomic sequences in about 5% of the mammalian cells, as detected by indirect immunofluorescence. One Ltk- cell in 300 to 500 was transformed to the TK+ phenotype after fusion with protoplasts carrying the chimeric plasmid pX1, which consists of pBR322 and the BamHI fragment coding for the herpes simplex virus type 1 thymidine kinase gene.
Authors:
R M Sandri-Goldin; A L Goldin; M Levine; J C Glorioso
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Publication Detail:
Type:  Journal Article; Research Support, U.S. Gov't, Non-P.H.S.; Research Support, U.S. Gov't, P.H.S.    
Journal Detail:
Title:  Molecular and cellular biology     Volume:  1     ISSN:  0270-7306     ISO Abbreviation:  Mol. Cell. Biol.     Publication Date:  1981 Aug 
Date Detail:
Created Date:  1997-09-26     Completed Date:  1997-09-26     Revised Date:  2009-11-19    
Medline Journal Info:
Nlm Unique ID:  8109087     Medline TA:  Mol Cell Biol     Country:  UNITED STATES    
Other Details:
Languages:  eng     Pagination:  743-52     Citation Subset:  IM    
Affiliation:
Department of Human Genetics, University of Michigan Medical School, Ann Arbor 48109, USA.
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MeSH Terms
Descriptor/Qualifier:
Animals
Cell Fusion*
Cell Wall / metabolism
Cercopithecus aethiops
Cloning, Molecular
Gene Transfer Techniques*
Genes, Viral*
In Situ Hybridization
Muramidase / metabolism
Plasmids / metabolism
Protoplasts / cytology*
Receptor Protein-Tyrosine Kinases / genetics
Receptors, Immunologic / genetics
Simplexvirus / genetics*
Vero Cells
Grant Support
ID/Acronym/Agency:
5P40-RR00200/RR/NCRR NIH HHS; F32-AI05653/AI/NIAID NIH HHS; T32-GM-07544/GM/NIGMS NIH HHS
Chemical
Reg. No./Substance:
0/Receptors, Immunologic; EC 2.7.10.1/Receptor Protein-Tyrosine Kinases; EC 3.2.1.17/Muramidase
Comments/Corrections

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