Efficient co-transfection of Primary rat cortical neurons at DIV 3 with plasmid DNA using NeuroMag (Magnetofection)

At 3 days in vitro (DIV), cultures of primary cortical neurons were co-transfected using NeuroMag at a 3.5:1 ratio (3.5 µL NeuroMag per µg DNA).

This paper shows the high efficiency of NeuroMag from OZ Biosciences to transfect primary cortical neurons at low DIV with two plasmid (co-transfection).

article reference: Mol Cell Neurosci. 2014 Aug 7;62C:42-50.

N-myristoylation regulates the axonal distribution of the Fragile X-related protein FXR2P.

Stackpole EE, Akins MR, Fallon JR.

AbstractFragile X syndrome, the leading cause of inherited intellectual disability and autism, is caused by loss of function of Fragile X mental retardation protein (FMRP). FMRP is an RNA binding protein that regulates local protein synthesis in the somatodendritic compartment. However, emerging evidence also indicates important roles for FMRP in axonal and presynaptic functions. In particular, FMRP and its homologue FXR2P localize axonally and presynaptically to discrete endogenous structures in the brain termed Fragile X granules (FXGs). FXR2P is a component of all FXGs and is necessary for the axonal and presynaptic localization of FMRP to these structures. We therefore sought to identify and characterize structural features of FXR2P that regulate its axonal localization. Sequence analysis reveals that FXR2P harbors a consensus N-terminal myristoylation sequence (MGXXXS) that is absent in FMRP. Using click chemistry with wild type and an unmyristoylatable G2A mutant we demonstrate that FXR2P is N-myristoylated on glycine 2, establishing it as a lipid-modified RNA binding protein. To investigate the role of FXR2P N-myristoylation in neurons we generated fluorescently tagged wild type and unmyristoylatable FXR2P (WT and G2A, respectively) and expressed them in primary cortical cultures. Both FXR2P^WT and FXR2P^G2A are expressed at equivalent overall levels and are capable of forming FMRP-containing axonal granules. However, FXR2P^WT granules are largely restricted to proximal axonal segments while granules formed with unmyristoylatable FXR2P^G2A are localized throughout the axonal arbor, including in growth cones. These studies indicate that N-terminal myristoylation of the RNA binding protein FXR2P regulates its localization within the axonal arbor. Moreover, since FMRP localization within axonal domains requires its association with FXR2P, these findings suggest that FXR2P lipid modification is a control point for the axonal and presynaptic distribution of FMRP.

NeuroMag is the first dedicated Magnetofection ™ transfection reagent for neurons. It is perfect for primary neurons and can also be used with cell lines and glial cells. The transfection can be performed with neurons from 3 DIV to 21 DIV. It can also be used on ES cell derived motor neurons.