4 replies to this topic

[eurisko]

hi, i am currently working with shRNA which is cloned into a pKLO plasmid vector with a U6 promoter. i transfect mammalian cells with this plasmid and i am currently aiming at getting stable clones.
but after 14 days of transfection most of my cells are dying and i am unable to isolate any clone.
how do i continue at this rate?
how do i confirm whether the death is occurring due to the silencing of the desired gene, and this gene expression is indispensable to the survival of my cells due to which the cells are unable to survive or is the death occuring because the shRNA is somehow toxic to my cells.
is there any assay i can do to confirm this?
please help

Edited by eurisko, 26 September 2010 - 10:52 AM.

[laurequillo]

Why dont you produce the virus? If your aim is to create stable clones I would infect the cell, better than transfect them. Anyway, Dont you have a control, like shScrarmble or ShLuc...something like that?

[eurisko]

Why dont you produce the virus? If your aim is to create stable clones I would infect the cell, better than transfect them. Anyway, Dont you have a control, like shScrarmble or ShLuc...something like that?

no i do not have the viral stocks. right now i have only the plasmid stocks in hand. can u suggest something that i can do with the plasmids stocks itself. and control too i still have to acquire.

[laurequillo]

Why dont you produce the virus? If your aim is to create stable clones I would infect the cell, better than transfect them. Anyway, Dont you have a control, like shScrarmble or ShLuc...something like that?

no i do not have the viral stocks. right now i have only the plasmid stocks in hand. can u suggest something that i can do with the plasmids stocks itself. and control too i still have to acquire.

so the first thing would be to get a control shRNA, shscramble, shGFP, shLuc...Or you could try using the empty vector as a control

Edited by laurequillo, 27 September 2010 - 01:40 AM.

[eurisko]

Why dont you produce the virus? If your aim is to create stable clones I would infect the cell, better than transfect them. Anyway, Dont you have a control, like shScrarmble or ShLuc...something like that?

no i do not have the viral stocks. right now i have only the plasmid stocks in hand. can u suggest something that i can do with the plasmids stocks itself. and control too i still have to acquire.

so the first thing would be to get a control shRNA, shscramble, shGFP, shLuc...Or you could try using the empty vector as a control

will get the vector soon.
but even then how do i isolate stable clones if my cells dont survive after the shRNA treatment. i uderstand that one of my questions can b answered on the treatment of of my cells side by side with a control vector but then what .
how do i attain the stable clones