I want to test the function of a protein by reducing a protein level in primary neurons. We used pSUPER by electrotransfection before, but it didn't work by western blot. In order to improve the effect， we want to use lentivirus infection. We obtained the pLKO.1 vector (hPGK promoter)from another lab, but we don't know whether it can be used in primary rat neurons.
By the way, is there any better method to do knockdown in primary neurons， especially for hippocampus and cortical neurons？ I would appreciate it if anyone could help me.
Thanks all,
Xu Yiwen
The pLKO.1 vector can deliver DNA to primary cortical neurons by packaging it with VSV-G envelope vector. And since the shRNA is expressed from U6 promoter of pLKO.1, even if puro expression from hPGK promoter is weak, knocking down may still be successful.
https://cdn.origene.com/assets/documents/lentiviral/does_lentivirus_work_with_neurons.pdf
Yohei Kono Thanks for your answer. Yes, we find that the plasmids work in PC12 but we can't detect the expression of mCherry (hPGK promoter). But in rat primary cortical neurons, we even didn't see any knocking down effect by transfecting the plasmids.
Whether only the PLKO.1 can work well as virus?
Are you just transfecting pLKO.1 plasmid without packaging it as a lentivirus?
PC12 is a strained cell line, so it is easily transfectable plasmids, but primary cells are completely different. Moreover, it is not surprising that mCherry is invisible because the promoter is easily methylated and silenced. Still, the U6 promoter works, so knockdown is possible, but as I guess, you haven't successfully transfected it at all. It is impossible to transfect a pLKO plasmid into primary cells unless you package the lentivirus.
Yohei Kono We want to screen for two effective shPOIs from 5 plasmids targeting different sequences before packaging the lentivirus, so we just tranfected the plasmids into PC12 or primary nuerons by electrotranfection.
You are right, electrotranfection may don't work.
Thanks for your suggestion, we will use lentivirus to infect primary neurons later.
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