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TRANSFECTION OF PRIMARY SCHWANN CELL CULTURES AND OF ORGANOTYPIC DORSAL ROOT GANGLIA CULTURES: PRELIMINARY RESULTS AND TECHNICAL CONSIDERATIONS
Author(s) -
Zerega B.,
Nobbio L.,
Paleari L.,
Levi G.,
Abbruzzese M.,
Torre G.C.,
Banchi L.,
Mancardi G.L.,
Sche A.
Publication year - 2000
Publication title -
journal of the peripheral nervous system
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1
H-Index - 67
eISSN - 1529-8027
pISSN - 1085-9489
DOI - 10.1046/j.1529-8027.2000.00513-60.x
Subject(s) - electroporation , transfection , myelin , biology , microbiology and biotechnology , schwann cell , immunostaining , lipofectamine , cell culture , immunology , gene , neuroscience , genetics , immunohistochemistry , vector (molecular biology) , central nervous system , recombinant dna
The most common forms of Charcot‐Marie‐Tooth (CMT) disease are due to duplication/deletion of the myelin protein 22 gene (PMP22); however, several patients harbor point mutations in PMP22 or in other myelin related proteins. To study the mechanism by which dominant point mutations affect myelin formation and maintenance, we are attempting to optimize gene transfer protocols into Schwann cells (SC) that will introduce expression constructs carrying mutated cDNA. Initial experiments were carried out on pure primary cultures of SC from sciatic nerves of newborn rats, co‐cultured with primary sensory neurons under conditions inducing myelination in vitro. Using the EGFP expression vector, which induces endogenous green fluorescence in the transfected cells, we have tested a number of gene transfer approaches, including electroporation and polycationic reagents (FuGene). Best results are obtained with the FuGene reagent (Boheringer) (up to 25%), while the electroporation is less efficient (5%–12%). However, the difficulties in selecting and expanding SC after transfection need to be overcome. Thus, we are optimizing gene transfer procedures on organotypic cultures of dorsal root ganglia from 15 day‐old‐rat embryos. SC and neurons are identified by their morphology; immunostaining with S100 and antibodies to phosphorlated neurophylaments are used to recognize, respectively, SC and axons. Using either Lipofectamine 2000 (GIBCO) and FuGene, polycationic reagents, SC are trasfected to levels of efficiency comparable or better than pure culture protocols. The same cultures maintained for 15 days after transfection showed extensive axonal growth and initial myelin formation. Scattered SC were still expressing the transgene at a visible level. These advances will likely help in understanding the role of specific genes in myelination and learning how their mutated forms lead to hereditary demyelinating neuropathies.