Nano-injection of plasmids


Plasmids are circular small molecules of DNA widely used in molecular biology and biotechnology for genetic engineering. This protocol provides information for a rapid and efficient plasmid injection into HeLa cells using the FluidFM® technology. 

MATERIALS

FluidFM® nanosyringe, 800nm aperture, 2N/m nominal stiffness

TE buffer (10mM Tris-HCl pH7.4, 0.25mM EDTA)

HeLa cells growing in a TC treated 6-well plate

pEGFP-C1 (4731bp) and pmCherryC1-TRIM21 (6083bp) plasmids

CO2 independent growth medium, supplemented with 2nM L-Glutamine, 10% FBS and penicillin/streptomycin

Lucifer Yellow (2mg/ml fresh solution in HEPES buffer) (optional)

 
Warning!

Make sure that all solutions (buffers and cell media) have been filtered through a 0.2µm pore before starting the experiment.

PREPARATION

Probe Coating

The surface of the FluidFM® nanosyringe needs to be hydrophobic to avoid cell adhesion. Therefore, a thin layer of a silicone based solution (Sigmacote®) is added to the probe, following the coating protocol. The probe can be coated and stored up to one week before its use.

Plasmid preparation

Plasmids must be endotoxin-free and eluted in filtered TE pH7.4 buffer. Plasmids that have been precipitated in ethanol and resuspended in TE buffer are not recommended, since any trace of residual alcohol might affect cell physiology and viability.
Prepare a solution containing 2.5E10 copies per microliter of each plasmid.  

Note

Adding a fluorescent compound such as Lucifer Yellow to the solution helps to monitor the injection process.     


Reservoir filling

Dispense 1µl of the plasmid solution into the probe reservoir according to the probe preparation protocol. In order to avoid the drying of the solution into the probe, it is strongly recommended to load the probe on the BOT as fast as possible after this step.

Cells preparation

In order to facilitate the injection procedure, it is recommended to seed the cells at least 6 hours before the experiment.

Note

Using medium without phenol red helps to obtain a better visualization of the injection of fluorescent compounds. Therefore, follow the following steps:

  • Just before the injection procedure, remove the cell culture medium.

  • Add 2 ml of CO2 independent growth medium supplemented with 2nM of L-Glutamine, 10% FBS and penicillin/streptomycin.

 

PROCEDURE

Prepare the system

Turn on the BOT, the microscope and the computer.

Initialize the software.

Load the probe into the probe holder in the right port of the BOT.


 

Load the TC-treated 6-well plate containing the cells in the left port of the BOT.

Run the Prepare System workflow.


Injection

Once the system is ready, run the Injection workflow.

Selection of the cells

Use one of the proposed tools to target the nucleus or the cytoplasm of the desired cell. When performing cytoplasm injection, it is strongly recommended to select a point close to the nucleus. Otherwise, the probe can not enter properly into the cell and the product would be released in the cell culture medium.

Set parameters for injection

When using HeLa cells on a TC-treated multiwell plate and following precisely this protocol, the parameters used should be the following:

ParameterValue
Setpoint1µN
Approach Speed1µm/s
Retraction Speed5µm/s
Pressure40mbar
Injection Time15s
Retraction Distance

20µm

Note
Optimization of the parameters needs to be adjusted when using other cell lines or culture dishes. 


Perform injection.



Examples of plasmid injection into cells. (A) Bright field observation of cells 24h after the injection. Cell morphology appears to be unaffected after the injection. Viability of the culture was assessed by propidium iodide staining and showed 95% cell viability. (B) 14 out of 20 injected cells show expression of GFP, both in nucleus and cytoplasm. (C) 8 out of those 20 injected cells also show expression of mCherry-TRIM21 in the cytoplasm. 

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