Transfection of plasmid DNA in primary hippocampal neurons.

Primary cells and especially hippocampal neurons are very difficult to transfect. Magnetofection? technology allows increasing drastically transfection efficiency on these kinds of cells. Magnetofection? exploits magnetic force exerted upon gene vectors associated with magnetic particles to drive the vectors towards, possibly even into, the target cells. In this manner, the complete applied vector dose gets concentrated on the cells within a few minutes so that 100% of the cells get in contact with a significant vector dose.

Cell co-culture

Dissect Hippocampi from E18 embryonic rat?s brain in a 3^?C pre cooled Ca²⁺ and Mg²⁺ free HBSS solution and then incubate with 0.25% of trypsin for 15 min at 37?C. Wash Hippocampi two times with the HBSS solution. Triturate tissue by using a fire polished Pasteur pipette. Wait two minutes until non-dissociated tissue goes to the bottom of the tube. Transfer dissociated cells into a new tube, add 2 mL of HBSS solution to the remaining non-dissociated clusters and push it through the Pasteur pipette until the complete dissociation of the tissue is observed. Transfer dissociated cells into the tube containing the first portion of the cells. Calculate the number of cells with a Malassez cell. Dilute the cells to the desired concentration with culture medium. Plate the neurons and glial cells at a density of 300,000 to 400,000 cells / mL in 35 mm dishes, with 2 mL of culture medium per dish. Culture medium contains MEM and supplements. A specific recipe is provided to OZ Biosciences customers upon request. This recipe allows obtaining higher transfection efficiencies.

Transfection protocol

1. Cells

Primary cells were seeded at 300,000 to 400,000 cells / mL in 35 mm dishes, with 2 mL of culture medium per dish. The cell density is a critical parameter to achieve good transfection with low toxicity; the suitable cell density will depend on the growth rate and the conditions of the cells; higher cell confluence is preferable than low cell density.

2. Day of Transfection

Cells have been transfected from 4 to 21 d.i.v (day in vitro). The best results were achieved with cells cultured for 4-5 days in vitro and assayed on the next day.

3. Complexes Preparation

The DNA solution and the Lipofectamine? 2000 reagent (Lipid from Invitrogen) should have an ambient temperature and be gently vortexed prior to use. Never use the cold reagent.

The rapid protocol is as simple as follows; the complexes are prepared in one tube.

a. In one tube add 300 ?L of serum free medium (such as OptiMEM)

b. Dilute 1 ?g of DNA in this tube.

c. Add 7 ?L of Lipofectamine? 2000.

d. Finally, before each use, vortex the tube of CombiMag reagent (Magnetofection? from OZ Biosciences) and add 1 ?L of CombiMag to the mixture and mix immediately by vigorous pipetting.

e. Incubate at room temperature 30 minutes.

4. Transfection

a. Add drop by drop the complexes solution to the cells growing and gently rock the dish.

b. Place the cell culture dishes upon the magnetic plate (OZ Biosciences) for 20 minutes at 37?C.

c. Perform a medium change just after the 20 minutes incubation time on magnetic plate while the culture plate remains positioned on the magnetic plate.

d. Incubate the cells at 37?C in a CO2 incubator under standard conditions until evaluation of transgene expression. Maximum transfection efficiency is observed after 48-72 h post-transfection.

It is noticeable that transfection efficiency of hippocampal neurons highly depends on cell culture preparation (OZ Biosciences provides more tips about preparation of neurons competent for transfection). Transfection of primary neurons with Lipofectamine? 2000 but without CombiMag leads to poor efficiency. Note also that this protocol can be adapted to many others primary cells.