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I've recently been performing shRNA transfections and was hoping to get some advice on why I'm not seeing any knockdown via western blot

I transfected by MDAMB468 breast cancer cell lines with shRNA against PTEN from the pLKO.1 lentiviral vector. I selected for the transfected cells using 1ug/ml puromycin for approximately 6 days. During this time there was very few cell death amongst the cells compared to my control plate (untransfected cells with 1ug/ml puromycin), suggesting that the transfection was successful.

After the 6 days I harvested my cells for protein and RNA extraction. I performed a western yesterday to assess knockdown, but noticed that there was hardly any differences (if any!) compared to my shGFP control. I haven't done a qpcr yet but I plan to this week.

I was wondering if anyone has experienced something similar, especially when working with PTEN? could it be that the cells need to be selected for a longer period (i.e. more than a week) before assessing protein knockdown?

Thanks!

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  • $\begingroup$ I don't know much about this protocol, but is it possible that you transfect enough for antibiotic resistance expression, but not enough for shRNA to actually work? $\endgroup$ – aaaaa says reinstate Monica Jul 12 '15 at 21:37
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I assume that you transfected the plasmid directly not producing viruses. If you want stable transformants, you might want to subculture them and maintain a bit longer. It is because efficiency of integration of plasmids into genome could not be that high--less than 0.1% of population obtain stable resistance by integration into genome. After 6 days, cells were still healthy in your case. This means you might get good transfection efficiency and after several times of cell division, your plasmids were diluted and the effects might be low even if cells maintain resistance against puromycin.

If transient expression (KD) works in your experiment, you might want to check expression level of your target at day 2 or 3. If you prefer stable cell lines, you can increase puromycin concentration, and if you agree, you pick up single colonies after cells not having plasmids in genome are dead. Among these colonies, some have more plasmid copies other have less. You can choose cells least expressing your targets as well as moderately expressing cells if you want. Because you might want to see expression level dependency.

For some cells, 5ug/ml puromycine is used according to this link.

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  • $\begingroup$ thanks for the suggestions. I don't have much experience with shRNA transfections, but do you think it's possible that the protein (PTEN) is quite stable and the cells would need to be cultured for longer (what would you recommend, 2 weeks?) to see the knockdown? I'm interested in doing the qPCR to see how the mRNA knockdown looks... $\endgroup$ – tolo9397 Jul 12 '15 at 23:05
  • $\begingroup$ @tolo9397 Try that qPCR. It is possible that your shRNA sequence is not binding like you wanted it to. Have you verified that this shRNA actually knocks down your gene of interest? $\endgroup$ – user137 Jul 13 '15 at 0:23
  • $\begingroup$ @tolo9397 As you mention, in general, KD effects could be delayed from a shRNA expression peak because of protein life time. So, cells may be cultured one or two more days compared with protein expression vector transfection. If the delay is more than that, you might want to transfect shRNA expression plasmid again several days after the first transfection to keep shRNA effects high. If you need stable transformants, this is not the case, though. $\endgroup$ – 243 Jul 13 '15 at 4:34
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    $\begingroup$ @tolo9397 PTEN is a popular protein, so you could find KD experiments published like nature.com/nm/journal/v17/n4/full/… $\endgroup$ – 243 Jul 13 '15 at 4:34

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