Can/should I linearize your viral vectors for stable integration?
Linearizing the DNA reduces the uptake into the cell, as circular DNA has a 4X greater chance of cellular uptake. However, once in the cell, linearized DNA does show better integration than circular DNA.
pLKO.1 - NcoI - based on our current map, this should cut just downstream of the polyA signal.
pGIPZ - FspI at base 10,755 (cuts inside ampicillin marker) NOTE: Was changed from the previously recommended PmeI since it cuts in the poly A signal which might not be good.
pTRIPZ - PmeI at position 8404 should work. This lies just downstream of the 3' LTR so it will cut the entire viral transcript (LTR to LTR).
pSMP** - NdeI at position 6149, which is outside the LTRs
pSM2** - ApaI at base 5055. It lies between the 3LTR and the RK6 promoter.
** Please note the pSM2 and pSMP libraries, constructs, gene sets and families and RNAintro kits have been discontinued.
Note: We do not linearize our vectors in house, but offer the above suggestions as to which sites to use. All these sites should linearize the respective plasmid in an area that is inconsequential to those components that function post integration into the mammalian genome. The maps used to determine these sites are of EMPTY vectors. Since each shRNA clone contains a unique target sequence as well as a unique barcode there is a small chance that any one clone could contain a second restriction site for the enzyme given. Therefore, when the digest is done a small aliquot should be run on a gel to ensure a single cut occurred, linearizing the vector.