10 replies to this topic

[deepgreenbegum]

Hi,

Does anybody have any experience with using a lentivirus to knockdown specific miRNAs in vitro? I am having a lot of trouble transfecting hard-to-transfect, suspension cell lines like HL-60s and NB4s using liposomes or electroporation (tried Amaxa and Neon to transfect LNA-oligos for knockdown, too).

I am thinking of buying a custom made lentivirus (maybe miRZip) and transducing the cells with that but I couldn't find any literature that has done this. Anybody has any opinions if that'd work? OR should I try something other than LNA-based miRNA inhibitors?

[Jon Moulton]

A Vivo-Morpholino should work for suspension cells.
http://www.gene-tool...vivomorpholinos
http://www.gene-tools.com/node/31
I work for the folks that make these oligos.

[Mucinman]

deepgreenbegum, on 02 November 2010 - 09:07 AM, said:

Hi,

Does anybody have any experience with using a lentivirus to knockdown specific miRNAs in vitro? I am having a lot of trouble transfecting hard-to-transfect, suspension cell lines like HL-60s and NB4s using liposomes or electroporation (tried Amaxa and Neon to transfect LNA-oligos for knockdown, too).

I am thinking of buying a custom made lentivirus (maybe miRZip) and transducing the cells with that but I couldn't find any literature that has done this. Anybody has any opinions if that'd work? OR should I try something other than LNA-based miRNA inhibitors?

I am interested in what your plans are. I am also trying to knockdown miRNA in a difficult to transfect cell line (HCE). I currently use the addgene protocol (psPax2, pMD2.G, and either pLKO.1 or pSicoR to clone into) to make my own virus for knockdowns. I am not sure what would go into making my own miRNA knockdowns though. Would it be as simple as a antisense sequence? How would I ensure it is cleaved properly?

[scolix]

Do you know if the lentivirus will infect and express in your cells? Some cells are difficult to expression even with lentivirus. Assess it carefully.
I have heard someone talk about knocking down miRNA using lenti but cannot remember it now.

[deepgreenbegum]

I am also planning to construct my own lentivirus for knockdown. I have produced lentivirus before but this timer we're going to be using H1 promoter. I've found this one article and they used pBluescript-H1 plasmid. Is this a commercially available plasmid or do I need to but pBluescript and clone H1 into it? How is your results coming along using the Addgeve protocol?

Mucinman, on 10 November 2010 - 01:21 PM, said:

deepgreenbegum, on 02 November 2010 - 09:07 AM, said:

Hi,

Does anybody have any experience with using a lentivirus to knockdown specific miRNAs in vitro? I am having a lot of trouble transfecting hard-to-transfect, suspension cell lines like HL-60s and NB4s using liposomes or electroporation (tried Amaxa and Neon to transfect LNA-oligos for knockdown, too).

I am thinking of buying a custom made lentivirus (maybe miRZip) and transducing the cells with that but I couldn't find any literature that has done this. Anybody has any opinions if that'd work? OR should I try something other than LNA-based miRNA inhibitors?

I am interested in what your plans are. I am also trying to knockdown miRNA in a difficult to transfect cell line (HCE). I currently use the addgene protocol (psPax2, pMD2.G, and either pLKO.1 or pSicoR to clone into) to make my own virus for knockdowns. I am not sure what would go into making my own miRNA knockdowns though. Would it be as simple as a antisense sequence? How would I ensure it is cleaved properly?

[deepgreenbegum]

I've read several papers where they used a lentivirus for miRNA knockdown in the cell lines that I'm using. It's quite complicated at the cloning step though...

scolix, on 11 November 2010 - 11:30 PM, said:

Do you know if the lentivirus will infect and express in your cells? Some cells are difficult to expression even with lentivirus. Assess it carefully.
I have heard someone talk about knocking down miRNA using lenti but cannot remember it now.

[UBClabbie]

Here are some links to already made lentivirus and adenovirus miRNA knockdown clones. Saves you time and frustration since cloning is the most annoying part.

http://www.abmgood.c...11838&dsn=12254
http://www.abmgood.c...11838&dsn=12255

[deepgreenbegum]

Thank you for these links! Ordering a virus from them is definitely an option, they're not too expensive.

biotechgirl, on 14 January 2011 - 12:51 PM, said:

Here are some links to already made lentivirus and adenovirus miRNA knockdown clones. Saves you time and frustration since cloning is the most annoying part.

http://www.abmgood.c...11838&dsn=12254
http://www.abmgood.c...11838&dsn=12255

[deepgreenbegum]

hi biotech girl,

have you used any of these vectors/viruses? Do they work OK? We may purchase a virus but need to decide which company to go with...

biotechgirl, on 14 January 2011 - 12:51 PM, said:

Here are some links to already made lentivirus and adenovirus miRNA knockdown clones. Saves you time and frustration since cloning is the most annoying part.

http://www.abmgood.c...11838&dsn=12254
http://www.abmgood.c...11838&dsn=12255

[UBClabbie]

deepgreenbegum, on 26 January 2011 - 09:49 AM, said:

hi biotech girl,

have you used any of these vectors/viruses? Do they work OK? We may purchase a virus but need to decide which company to go with...

biotechgirl, on 14 January 2011 - 12:51 PM, said:

Here are some links to already made lentivirus and adenovirus miRNA knockdown clones. Saves you time and frustration since cloning is the most annoying part.

http://www.abmgood.c...11838&dsn=12254
http://www.abmgood.c...11838&dsn=12255

I haven't used them personally but know lots of people who have and had great results. Don't let the price fool you, they're just as good as the bigger name competitors. My suggestion would be to call them or email them if you have any questions about quality. I know they have a guarantee on their products and try really hard to help you troubleshoot if something doesn't work. There's a big sale on miRNA related stuff right now too and lenti is prob included

Good luck with your experiment. Let me know how it goes if you decide to order from them.

[Earnest]

biotechgirl, on 26 January 2011 - 04:45 PM, said:

deepgreenbegum, on 26 January 2011 - 09:49 AM, said:

hi biotech girl,

have you used any of these vectors/viruses? Do they work OK? We may purchase a virus but need to decide which company to go with...

biotechgirl, on 14 January 2011 - 12:51 PM, said:

Here are some links to already made lentivirus and adenovirus miRNA knockdown clones. Saves you time and frustration since cloning is the most annoying part.

http://www.abmgood.c...11838&dsn=12254
http://www.abmgood.c...11838&dsn=12255

I haven't used them personally but know lots of people who have and had great results. Don't let the price fool you, they're just as good as the bigger name competitors. My suggestion would be to call them or email them if you have any questions about quality. I know they have a guarantee on their products and try really hard to help you troubleshoot if something doesn't work. There's a big sale on miRNA related stuff right now too and lenti is prob included

Good luck with your experiment. Let me know how it goes if you decide to order from them.

Chiming in on the had great results.  Our publication is coming out in half the time and cost cause we bought the product instead of the supplies to try cloning ourselves.  Sometimes, you just need someone who does it day in and day out to help solve the problem.