miR30 based shRNA in mouse cells - (Mar/15/2014 )
Hello everyone!
I’m using a miR30 based shRNA lentiviral system, namely the vectors pGIPZ, pCMV-dR8.91 and pCMV-VSV-G, in order to knockdown BV2 and N2a mouse cell lines. My current results are not encouraging since I saw no decrease of the gene expression using Real-time PCR and more important no GFP is detectable with fluorescent microscope after 4 weeks of culture with the antibiotic of choice, puromycin. However the cells maintain their resistance against puromycin. So my questions are:
Does anyone know if all of these vectors are compatible with mouse cell lines and has anyone used them successfully before to knockdown mouse cells?
In a precedent post it was mentioned that GFP expression is diminished when the shRNA is being effectively processed due to their existence in the same transcript (http://www.protocol-online.org/forums/topic/9039-why-the-fluoresence-is-dim-or-dispear-in-mirna-vector/). Wouldn’t that cause loss of resistance too? Admittedly I lowered the antibiotic concentration to help in raising individual cell clones. Could this have caused loss of viral vectors gene expression?
Two things:
Could you detect GFP earlier after infection?
Maintain selection until you are sure that the genes are integrated. Lowering before they are could easily result in loss of the transgene.
I'm not familiar with the plasmids, but in general antibiotic selection is not from the same transcript as the stuff found around the cloning site, it is usually from a separate ORF.
bob1 on Sat Mar 15 23:38:09 2014 said:
Two things:
Could you detect GFP earlier after infection?
Maintain selection until you are sure that the genes are integrated. Lowering before they are could easily result in loss of the transgene.
I'm not familiar with the plasmids, but in general antibiotic selection is not from the same transcript as the stuff found around the cloning site, it is usually from a separate ORF.
bob1 I neglected to check regularly for GFP immediately after infection, something that won't happen again, mainly because I relied too much on the antibiotic resistance. However I saw GFP after transfection of the plasmids to the packaging cell line HEK 293FT so I know that the virus was produced.
Nevertheless 293FT is a human cell line so my question about the compatibility of the aforementioned plasmids towards mouse cells still stands! I would really like to know if anyone has used this combination of plasmids in a successfull lentiviral system for knockdown in mouse cell lines.
Indeed I dropped puromycin concentration too much, even according to my cytotoxicity curve, so this time I'll keep the concentration high and check regularly for GFP.
I assure you that GFP, puromycin resistance gene and shRNA exist on the same transcript and the first two are connected with an IRES.
Wouldn't the presence of GFP in your packaging step just indicate that the GFP plasmid is functioning - it doesn't necessarily indicate that you got virus out, unless you then purified the virus and titrated it somehow to tell you that you have active virus.
Kostas on Sun Mar 16 19:40:20 2014 said:
In general plasmids that will work in humans will also work in mice, this is because we use the same promoters (usually viral) for the production of the mRNA off the plasmid.However I saw GFP after transfection of the plasmids to the packaging cell line HEK 293FT so I know that the virus was produced.
Nevertheless 293FT is a human cell line so my question about the compatibility of the aforementioned plasmids towards mouse cells still stands! I would really like to know if anyone has used this combination of plasmids in a successfull lentiviral system for knockdown in mouse cell lines.
Kostas on Sun Mar 16 19:40:20 2014 said:
If it is structured GFP-IRES-PURr-shRNA, then the IRES would separate the production of GFP from the others so it shouldn't be tied to the production of the shRNA.I assure you that GFP, puromycin resistance gene and shRNA exist on the same transcript and the first two are connected with an IRES.