Lipopolyamine‐Mediated Transfection Allows Gene Expression Studies in Primary Neuronal Cells

Abstract

A simple and efficient transfection technique based on lipopolyamine-coated DNA that can be used for gene transfer in cerebellar granular neurons is described. Gene transfer is achieved by exposure of cells to a DNA/lipid complex obtained by simple mixing of lipopolyamine and plasmid DNA. This procedure may represent a general tool of physiological investigations in primary cells. We show that the promoters of the introduced chimera genes are regulated by their respective trans-acting factors and may be modulated via membrane receptors and second messengers. This procedure has no noticeable toxic effects, nor does it seem to interfere with complex physiological behavior like neuronal differentiation.

Synthetic vectors for gene delivery: An overview of their evolution depending on routes of administration.

“…Finally, it is worth noting the importance of spacer polycationic lipid derivatives of spermine. Indeed, as the polar head binds to DNA at the minor groove of the double helix, the linker needs to be long enough to avoid steric hindrance between the DNA and the hydrophobic cluster of the cationic lipid [29]. Polycationic lipids that utilize a polar head similar to natural aminoglycoside compounds have also been synthesized.…”

“…It is characterized by a polar head of spermine, which is connected through a spacer motif that includes a metabolizable peptide bond between two chains of saturated C18 fatty acids. The polycationic lipid (DOGS /DOPE) is marketed under the name Transfectam ® (Promega), and allows high transfection efficiencies in eukaryotic cells, due to the fusogenic properties of DOPE [29]. Even in the absence of a colipid, DOGS is effective.…”

Characterisation of wild‐type and mutant forms of human monoamine oxidase A and B expressed in a mammalian cell line

“…Villeneuve-La Garenne, France) per ml, for 2 h at 37°C under 5% CO, [9]. The cells were grown for another 48 h in MEM+ and then collected, washed twice with PBS, pH 7.4, and frozen as a cell pellet, Transfection was performed with the native MAO-A and -B constructs, both in sense and antisense orientation (mock-transfection) to the promoter, and with the chimera or mutated constructs described above.…”

Concurrent Measurement of Promoter Activity and Transfection Efficiency Using a New Reporter Vector Containing both Photinus pyralis and Renilla reniformis Luciferase Genes

“…Also, site-directed mutation can be performed in pJDL cmv reporter vector with the GeneEditor mutagenesis kits. Because all transfection experiments were performed with the calcium phosphate method, similar experiments were performed to verify the aforementioned data by different transfection methods such as DEAEdextran and cationic lipid transfection agents (15)(16)(17). In all three transfection methods, the values of promoter activity normalized with transfection efficiency were similar in HeLa and NT cells (data not shown).…”