Multi-lipofection efficiently transfected genes into astrocytes in primaryculture

This study demonstrated that liposome-mediated transfection - lipofection - is suitable for delivering genes into astrocytes. By repeatedly lipofectin g the same astrocyte cultures, a process we call multi-lipofection, the tra nsfection efficiency of the beta -galactosidase (beta -gal) gene was improv ed from 2.6+/-0.6 to 17.4+/-1.1%. This is the highest efficiency ever repor ted in gene-transfer with Lipofectin(R) in a primary culture of mouse cereb ral cortical astrocytes. Furthermore, multi-lipofection did not cause obser vable disturbance to astrocytes as indicated by insignificant changes in th e glial fibrillary acidic protein content in the cultures. In order to demo nstrate that the transfected gene achieved a physiologically relevant expre ssion level, a plasmid containing the pEF-hsp70 protein gene was lipofected into astrocytes. This produced colonies of astrocytes showing an increased resistance to heat-induced cell death. A similar experiment was performed with the glial-derived neurotrophic factor (GDNF) gene. Control astrocytes had no detectable GDNF. In the transfected astrocytes, the GDNF protein cou ld be identified intracellularly by immunocytochemistry. Western blot analy sis revealed, as compared to astrocytes with one lipofection, a 2.9-fold in crease of GDNF with four lipofections. GDNF remained detectable in astrocyt es 2 weeks after four lipofections. Thus, multi-lipofection provides a mild and efficient means of delivering foreign genes into astrocytes in a prima ry culture, making astrocytes good candidate vehicle cells for gene/cell th erapy in the CNS. (C) 2000 Published by Elsevier Science B.V.