26 November 2016 0 9K Report

Hi guys,

Recently, I try to knockdown two genes in MEF cell by using tet-induced knockdown lentivirus provided by this paper (attached). several steps are involved.

1. Cut the plasmid Tet-pLKO-Neo (Plasmid #21916) with AgeI and EcoRI. and insert my shRNA primer (annealled). Finally I get the correct sequence with my insert shRNA.

2. do lentivirus packaging and use the virus to infect MEF cell.

3. do G418 selection (800ug/ml) 2 days post-infection. change medium with G418 per two days.

4. after 4 days, all the negative control cells die.

5. subculture them (Scr, ACO-shRNA, and PexR-shRNA) into 24 well plates.

6. treat them with 0, 1, 3, 6, 12ug/ml doxycycline for 4 days (change medium with doxycycline per two days).

7. collect samples at day 4 and do qPCR. no any reduction for mRNA level for these two genes compared to Scr cells.

My labmate try Scr and ACO-shRNA in ins-1 cells. it worked well. 

The shRNA sequence used are the same as regular lentivirus plasmid (puro-lentivirus, non-inducible, addgene number: #1864), and they show really high efficiency. U6 promoter was used in regular plasmid, but H1 promoter was used in Tet-pLKO-Neo. So H1 promoter didn't work well in MEF cell? I hope guys can give me some advice. Thanks a lot.

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