Transfection of primary rat cortical neurons using the Magnetofectamine Kit

Primary neuronal cultures of cerebral cortex prepared from rat embryos were transfected with plasmid DNA using the Magnetofectamine Kit after 12 to 17 days in culture.

This paper demonstrated the high efficiency of Magnetofectamine from OZ Biosciences to transfect plasmid DNA into primary cortical cultures.

article reference: J Biol Chem. 2013 Nov 29;288(48):34897-905. 

Endoplasmic Reticulum-associated Degradation Controls Cell Surface Expression of γ-Aminobutyric Acid, Type B Receptors.

Zemoura K, Schenkel M, Acuña MA, Yévenes GE, Zeilhofer HU, Benke D.

Abstract

Metabotropic GABAB receptors are crucial for controlling the excitability of neurons by mediating slow inhibition in the CNS. The strength of receptor signaling depends on the number of cell surface receptors, which is thought to be regulated by trafficking and degradation mechanisms. Although the mechanisms of GABAB receptor trafficking are studied to some extent, it is currently unclear whether receptor degradation actively controls the number of GABAB receptors available for signaling. Here we tested the hypothesis that proteasomal degradation contributes to the regulation of GABAB receptor expression levels. Blocking proteasomal activity in cultured cortical neurons considerably enhanced total and cell surface expression of GABAB receptors, indicating the constitutive degradation of the receptors by proteasomes. Proteasomal degradation required Lys(48)-linked polyubiquitination of lysines 767/771 in the C-terminal domain of the GABAB2 subunit. Inactivation of these ubiquitination sites increased receptor levels and GABAB receptor signaling in neurons. Proteasomal degradation was mediated by endoplasmic reticulum-associated degradation (ERAD) as shown by the accumulation of receptors in the endoplasmic reticulum upon inhibition of proteasomes, by the increase of receptor levels, as well as receptor signaling upon blocking ERAD function, and by the interaction of GABAB receptors with the essential ERAD components Hrd1 and p97. In conclusion, the data support a model in which the fraction of GABAB receptors available for plasma membrane trafficking is regulated by degradation via the ERAD machinery. Thus, modulation of ERAD activity by changes in physiological conditions may represent a mechanism to adjust receptor numbers and thereby signaling strength.

CombiMag from OZ Biosciences composing the Magnetofectamine Kit is a very efficient transfection reagent based on Magnetofection™ technology.