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We use a lentivirus packaging vector called pLenti_puro_DEST. We use puromycin to select for the cloned gene in 293T cells. However, the pLenti_Neo_Dest has been discontinued, so I'm cloning a Neomycin resistance gene into the pLenti_Puro_Dest thus replacing Puromycin with Neomycin. I usually just grow up this gene in Stb3 or Stb4 cells from invitrogen. I'm wondering if I can select for cloned pDEST vectors in these cells using Neomycin or G418. According to the G418 manual, you can theoretically use it for bacterial selection, but I think that may be for libraries and not actually growing up the plasmid.

So is it possible to select for my plasmid in these cells by adding a little G418 to the ampicillin plate? Is this common? I can't find anyone selecting bacteria by Neomycin anywhere.

EDIT - The main reason I ask, is that the resistance gene is under a PGK promoter that I have no idea if it works in bacterial cells.

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  • $\begingroup$ Mammalian promoters don't work in bacteria basically. $\endgroup$
    – 243
    Jun 12, 2015 at 2:14

2 Answers 2

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PGK is mammalian (murine) phosphoglycerate kinase promoter. It will therefore not work in bacterial systems. Why don't you select using ampicillin only? You can increase the ampicillin concentration if you want.

Regarding:

I can't find anyone selecting bacteria by Neomycin anywhere.

Kanamycin is generally used for bacterial selection. The Neomycin resistance gene works on Neomycin, Kanamycin and G418 (a gentamycin variant). The gene basically codes for an aminoglycoside phosphotransferase and different variants of the gene have different affinities for different aminoglycoside substrates. For a basic reference see this.

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  • $\begingroup$ I do select with ampicillin, but any residual parent vector (from the cloning) would also contain ampicillin. $\endgroup$
    – jwillis0720
    Jun 12, 2015 at 6:08
  • $\begingroup$ @jwillis0720 For that you have to do screening. You can do colony-PCR screening which is relatively faster than other methods. Also have self ligation controls to have an idea of how many colonies you need to screen. This is sticky end cloning right? $\endgroup$
    – WYSIWYG
    Jun 12, 2015 at 8:08
  • $\begingroup$ Plasmid construction is a technique to get plasmids that carry your inserts from empty vectors carrying the same resistant markers. If you want to select with kanamycin/neomycin, you could take a bacterial expression unit of a kanamycin/neomycin gene. In fact, there are plasmids which carry kanamycin with both bacterial and mammalian promoters, $\endgroup$
    – 243
    Jun 12, 2015 at 14:42
  • $\begingroup$ I will just screen by PCR. But it's gibson assembly. $\endgroup$
    – jwillis0720
    Jun 12, 2015 at 20:18
  • $\begingroup$ @jwillis0720 not an issue. Just use the end primers and screen for the appropriately sized amplicon. $\endgroup$
    – WYSIWYG
    Jun 12, 2015 at 20:23
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In this paper (Li and Elledge, Nature Genetics, 2005) it is shown that the PGK promoter works in E.coli but it has very little activity. So, I don't think it is suited to properly express resistance enzyme.

About Neomycin, many aminoglycosides resistance enzymes (not all) that work on Kanamycin works well also with Neomycin. I have personally selected E.coli expressing Kan resistance (from a pET vector) using Neomycin without problems.

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