We have made two sets of vectors for DamID in mammalian cells: one set for conventional transfections, and one set for lentiviral transductions. These vectors differ somewhat in their design and applications.
DamID in mammalian cells in principle works with transient transfections. However, plasmids can accumulate in large amounts in cells after transient transfection. Since these plasmids are typically adenine-methylated, they can use up a large part of the sequencing reads. For this reason, we recommend that you either use stably transfected cells or lentivirus transduction for DamID.
Plasmids for conventional transfection (set A)
These vectors are derived from pIND/V5-HisA (originally from Invitrogen, but to our knowledge no longer commercially available). Expression is driven by an ecdysone-inducible promoter. For DamID we only use the leaky expression from the uninduced promoter. If you want to check the Dam(fusion) protein by Western blotting or immunofluorescence microscopy, expression must be induced by cotransfection of the activator plasmid pVgRXR (which we will also provide) and treatment with an ecdysone analog such as Ponasterone A (available from Invitrogen). The pIND backbone contains the neomycin selectable marker for making stable cell lines.
Sequences of the vectors for conventional transfections are available here.
Plasmids for lentiviral infections (set B)
Lentivirus transductions are quite suitable for DamID, even though it takes some work to make the virus preps. A major advantage is that lentiviruses give high transduction efficiencies in almost any cell type. Our vectors are derived from Inder Verma’s self-inactivating lentivirus system. The vectors are Gateway compatible, which means that you need to use GateWay recombination cloning to create a Dam-fusion of your protein. Many ORFs are now available as GateWay entry vectors from several sources. Otherwise you should make your own GateWay entry vector first.
The lentiviral vectors can be used in two ways. When transfected as a conventional plasmid, the strong viral promoter drives high expression of the Dam(fusion) proteins. This should not be used for DamID experiments, but it is useful for checking of the proteins by Western blotting or immunofluorescence microscopy. When used as lentivirus, the strong promoter is deleted upon integration of the virus, and expression of the Dam(fusion) is now driven by the weak pIND-derived heatshock promoter.
Sequences of the lentiviral vectors are available here.
How to obtain DamID vectors
Please decide which vector sets you would like to use (set A or set B). The vectors can then be requested by sending an email to Bas van Steensel. You will then receive by email a Material Transfer Agreement, to be signed by yourself and the responsible official of your institute. As soon as we have received the signed MTA, we will ship the vectors by regular mail (FedEx takes too much paperwork, sorry).