Help! GFP transfection - (Jan/25/2007 )
Hi everybody. I wanna transfect the primary hippocampal neurons with GFP, but it is technically said to be challenging. If someone with such experiences could give me some suggestion or protocols? Thanks in advance.
---- frog @_@
It is very challenging to transfect neurons. Someone in the forum ( kathy) wrote that PEI could be used to transfect neurons - 9% efficiency.
You could read and try something like that.
I have only used viruses to transfect neuronal cultures.
Good Luck !!!
Scolix, thanx for the reply on my thread (sorry didnt comment there).
Frog,i am still trying to get more neurons positive using PEI (i only got one till now) 
i see some very faint fluorescence but didnt see any neurites positive though. I am trying different incubation times etc. my advice if you see weak fluorescence try to stain with GFP antibody maybe it is better than just fluorescence. i will try that too.
Scolix, did your neurons live after virus? here in the lab they all have terrible experiences with viruses. 
Frog,i am still trying to get more neurons positive using PEI (i only got one till now)
i see some very faint fluorescence but didnt see any neurites positive though. I am trying different incubation times etc. my advice if you see weak fluorescence try to stain with GFP antibody maybe it is better than just fluorescence. i will try that too. Scolix, did your neurons live after virus? here in the lab they all have terrible experiences with viruses.
The neurons do very well even after viral infection. Never had problems unless the virus prep is contaminated with other cellular debris which might cause cell death. But if the preps are clean, there should be no problem.
I have used 4 different viruses on neurons. 3 of them, never had problem but with one of them, the virus is toxic for the cells so kills them, any cell for that matter.
I want to give a try on several transfection agents on hand made by myself for the neurons. How do you guys isolate/culture primary neurons?
genehunter,
You will need pregnant mice or rats. Take out the embryos and dissect the brain region of interest. And there you have primary neurons. (very simplified explanation)
If you need a full protocol, let me know, i can send you mine but you might need someone to help with dissection. It can be tricky.
Scolix, I have access to pregnant mice. Could you send me the protocol? Thanks a lot. If I find any one of those that works for neurons, I'd be happy to share it with the members who are working on primary neurons. Do you have any suggestions on neuronal cell lines as alternative?
For neuronal cell lines, you could try SHSY5Y cells or PC12 cells. SHSY5Y are quite stubborn to transfect with calcium phosphate.
Give me a few days till I type the protocol, I only have a hard copy of it.
we use new born or postnatal mice here to prepare SCG neurons for transfection. embryonic mice will mean we have to kill the mother and this is not an option here 
However, I do think that PEI is not very effecient for SCG. i cant get repeatable results and dont like it. but to transfect with virus i need to clone my gene again into the virus genome right?
However, I do think that PEI is not very effecient for SCG.
i cant get repeatable results and dont like it. but to transfect with virus i need to clone my gene again into the virus genome right? Kathy,
You will need to clone your gene into a viral vector and you will have to make the virus, it might not be the best option for you if you wish to complete your project within a short time (within a month).