Dear Chenglong Yu,

I have been using electroporation for transfection for a long time. I would not recommend it, expecially for primary neurons. The amount of DNA required is at least 10 times that used with other transfection methods and also the number of cells is because they suffer a lot. However, I  do not know the rationale you have for choosing electroporation, so maybe you can explain. And I do not have a protocol to suggest, sorry,  that you must find yourself in any case. In my case, it was very important  to perform the elctroporation in physiological saline or PBS and not in culture medium, but I did not use neurons.

Chenglong,

Carla is right, cortical neurons are extremely difficult to electroporate because they do not divide. Most people forget that there are at least two membranes to pass through to get inside the nucleus. Your standard electroporation approach will only get your DNA into the cytoplasm, the second phase requires a breakdown of nuclear envelope during cell division. With that said, Lonza seems to have figured a way around that hurdle with their proprietary technology, The Nucleofector™. That platform is the only one that I know of and have used thus far (there may be other that I'm not aware of) that results in some measurable success in electroporating cortical neurons in culture. I would check them and their competitors out if you still want to electroporate cultured cortical neurons.

Dear Carla, 

Thank you for your suggestion. I have to use electroporation as I am doing some co-culture. And I want to transfect only one kind of neurons. I wonder do they need DNA that are Endotoxin free? Besides, will antibiotics in the medium affect? Is it because of FBS inside the culture medium that you do not use it?

Thank you.

Dear Lawrence,

Thank you for your information. Please help me with Lonza. But they did not show detailed protocol, only the No. of protocol recommended. I will really appreciate if you can give me some detail information about their Nucleofector protocol.

Besides, I have a naive question. As Carla said, neurons will suffer a lot after electroporation. I wonder will some unknown effect that will keep affect these survival neurons. I want to check neuronal transmission at ~DIV 14. Do you think the neurons are recovered at that time if their morphology looks OK? Shall I compare the electrophysiology (Maybe patch) for both transfected and untransfected neurons?

Another related question. As you mentioned it is difficult to deliver DNA into nuclear of neurons, will these transient transfected DNA lasted for 2 weeks? As you know, I want to check synaptic transmission at DIV 14. Is it too long?

Thank you anyway.

Chenglong,

Lonza sells their proprietary technology as a kit and so they will not disclose the exact formulation of their kits nor electrical pulse protocol used to successfully electroporate neurons in vitro. I'm afraid you'll have to buy their kits and borrow their electroporator from a colleague. 

As for recovery of neurons in vitro, ones that survive the electroporation recover within a day if not hours. The survival of neurons depends on your electroporation protocol. As a rule of thumb, you are always balancing pore size and open duration when electroporating any cell. The longer and wider you open the pores with an electric field the more DNA you can introduce in the cell but run the risk of membrane not able to close the pores afterwards. Thus, if your neurons survive the electroporation ordeal, you can do morphometry and ephys on them in a couple of days for sure (you need to give them time to grow processes).

Since neurons don't divide, the foreign DNA you put into the cell will last as long as the cell lives. There is no dilution effect with neurons. However, if the foreign DNA doesn't get into the nucleus it will just sit in the cytoplasm and do nothing. Remember, transcription occurs in the nucleus. 

Best of luck,

L

Chenglong,

I have always been told that it is better to use endotoxin-free DNA for neuronal transfection, but I work in vivo.  The people in my Department use either the Qiagen or Macherey-Nagel kits, but there lots of other companies out there that make them.

You might try adapting the cortical neuron in vivo iontoporation protocol of De La Rossa and Jabaudon from Nature Protocols.  It has additional chemical components that help the DNA get into the nucleus:

http://www.ncbi.nlm.nih.gov/pubmed/25474030

If you contact them, they will send you a protocol with additional hints.

Good Luck-

Jill

Hi Chenglong,

I just stumbled into this discussion. I never used electroporation but lipofection for transfecting rat cortical neurons (lipofectamine works wonderfully). Out of interest, can you explain to me how you achieve that only one cell type of your co-colture is transfected?

Thanks in advance, Laura

 Dear Lawrence,

Thank you. Sorry for the late reply, we are suffering typhoon these days. 

I am not sure that whether I fully understand. Do you mean that once neurons survived, they will recover after enough time? As for electroporation, I transfect neurons once after harvesting, and I will take images after DIV14. Is it long enough for me to escape those morphometry and ephys tests and suppose these neurons are healthy?

About the second part, does it mean that once I got some expression of the transfected gene, I can expect continuously expression regardless of time?

Thank you anyway.

Hi Jill,

Thank you so much for the information. I am very interested in the chemical components that help DNA pass through. I will read it carefully. 

Besides, can you give me the Cat# of Qiagen kit for Endotoxin free plasmid?

Thanks a lot.

Hi Laura,

Sorry for the late reply. I do not know about lipofection, I hope I could achieve wonderful transfection as you. 

Let me briefly explain. As using electroporation, transfection will be carried out immediately after harvesting neurons. Therefore, we can transfect different plasmid into different neurons or cells before plating.

I find a Jove video about similar strategy, which may explain better to you. Here is the linkage: http://www.jove.com/video/52408/an-optogenetic-approach-for-assessing-formation-neuronal-connections

Hope this will help.

Thanks, that's pretty cool!

Chenglong,

Sorry for the delay in my reply. I've been busy with experiments and paper.

To answer your questions in the order they were asked: 1) if your neurons survive the harsh conditions of trituration and electroporation, they will start to send out neurites in 24 hours if not overnight. No dying or dead neuron will send out processes, so I would call those survivors and yes they will grow in size and neurite complexity. We typically see GFP expression from electroporated vectors in 24 hrs. However, how long your fluorescence persists depends on your plasmid vector and half life of the fluorescent protein. We use CAG driven plasmids for life-long expression of fluorescent protein. In our hands, CMV driven expression doesn't last very long, a couple of days at best. If you want to see spine, DIV 14-21 should be long enough for your cultured neurons to send them out and long enough for some to mature into stubby or mushroom shaped ones, if you have a healthy culturing system. 2) Since your neurons don't divide, the number of plasmid in your transfected neurons should not change, in theory. However, whether and for how long the expression persists depends on the promoter used in constructing your plasmid. For indefinite continuous expression, I recommend CAG promoter not CMV.

best of luck,

L

Hi Chenglong,

I just noticed that you asked for the catalog number of the Qiagen kit.  The product is called the EndoFree Plasmid kit, the catalog number depends on the size of the prep you are purifying.  The one I used is kit 12362.

Here's hoping that some of the hints above were useful.

Jill

Hi Lawrence,

Thank you for the detailed answer. I understand now. I will try more. Recently, the first two trials shows 60~70% death of neurons after electroporation. I will keep work on it. There are some GFP at DIV3 and DIV5. I will keep test its expression on DIV14. 

Thank you for your kind help.

Hi Jill,

Thank you for the information. I wll try EndoFree kit this time. 

Anyway, thank you for your help these days. Hope I can achieve stable transfection.

Yu

Hi Chenglong,

I have a very similar situation like you. I am struggling to transfect cortical neuron ( I need to transfect before plating ). What is the best transfetion method you figured out finally ? Please let me know your opinion

Hi Riyadh,

Electroporation works fine with me. Although virus is also good, I think you can try electroporation before plating first.

Best,

Yu

Dear Chenglong Yu ,

I just stumbled upon your discussion here regarding the electroporation of neurons. I want to try the same but havent found any protocols, could you possibly share the protocol that you used?

Thank you,

Linn

Hi Linn,

Sure. I am using the elecporator from biored, which allowing the users to edit their own protocols. I am using the biopolor waveform with 2 peaks of 330V, and the inter-stimulation interval is 5ms. The peak lasts for 0.3ms for stimulation.

But I would like to suggest to modify according to your own neurons, and the different chamber you are using. It works for my striatal neurons.

Good luck.

Yu

Thanks a lot Chenglong Yu

I will give it a try.

/Linn

Hello Chenglong Yu,

I would like to use the Bio-rad device to do the transfection. However, there is not much information I could find.

I found you are using the same device as me, and you are doing quite well. May I have your precise protocol as the reference? Thanks a lot!

Tingting

Sorry I didn't mention, I would like to use rat hippocampal neuron, do you have experience with that kind of neurons?

Thank you so much for your advice.

Hi tinging,

I do tried rat hippocampal neurons before jumping to mice. Youe can find my protocol in this answers, as when I replying to Linn. I think hippocampal neurons are easier than cortical and much better than striatal neurons. If you have more questions, welcome to email me.

Good luck!