Anyone using "the hook"?
bernard at elsie.nci.nih.gov
Sun Jun 2 19:56:55 EST 1996
In article <31MAY96.19034119 at shrsys.hslc.org>, jovermeyer at shrsys.hslc.org
>I am considering trying Invitrogen's new transfection kit using a phook
>vector that entails magnetic beads on the cell surface to help select for a
>trsnsfected population of cells. Does anyone have any experience with this?
I am currently in the process of playing with this. Our current
"gold standard" for post transfection selection is an expression vector
for the human IL2 receptor and we can then capture the cells with
anti-IL2R-mab coated Dynal beads. This system works extremely well but
we want to sort cells that already have IL2R so I am trying this
Whilst the overall theory is sound the kit seems to have been
put together by Homer Simpson. It does work but needs some fiddling.
I've used the pHook-1 system (does not contain a MCS in the vector).
Here are some thoughts and tips;
1) Dump the pCR3LacZ test vector that comes with the kit.
It gives the lowest LacZ expression of any vector I have seen.
It also uses the same promoter (CMV) as the pHook plasmid (doh!)
so they would probably interfere with each other. I've used
the MacGregor pRSVZ or one of the SV derivatives (pSVbeta or
pCH110) and seen nice in situ staining of sorted cells.
2) They put a neo/kan cassette in the vector which already has
an amp cassette (doh!). This is entirely pointless as if you are
going for a stable line you'll use pSV2neo at the start and not
bother with the sorting and if you are sorting cells for transient
transfection you don't need another selectable marker. That's
0.8 kb of DNA wasted guys!
3) Invitrogen obviously hope to make most of their cash from
the PhOX beads which are obscenely over-priced, even by their own
standards. Look at the price of Dynal beads and do the maths to
check the mark-up. Alternatives to selling major organs to pay
for this are to use standard MACS with an antibody against either
the HA or myc epitope (I haven't tried this) or to make your own
derivatised beads. Since the hapten for their sFv is cheap and
spontaneously amine-reactive it should be possible to either react
it with protein coated beads (eg. an irrelavant antibody) or to
react it with a diamino spacer reagent (eg. diaminooctane) and thence
with tosylated dynabeads. I shall let you know how this works out.
4) The kit does not include a magnet (doh!) so get a good one.
5) If you've ever done any kind of MACS before using the kit
is a breeze. The one major practical problem is that Invitrogen
warn you against using trypsin to harvest cell prior to sorting
and suggest dispersion in PBS/EDTA. Since many adherent cell lines
just shrug off such treatment this results in unsortable clumps.
The IL2R system I use has the advantage that the surface antigen is
trypsin resistant so I can make nice single-cell suspensions for
sorting. Thankfully preliminary results with the Capture-Tec kit
suggest that Invitrogen are being over-cautious and that light
exposure of transfected cells to trypsin does not stop them from
being sorted. I'll try and confirm this next time.
6) I have no idea just how high the affinity of the antibody
is for the beads. Since the system is designed for positive
selection, rather than depletion, this only affects the yield.
I aim to perform a selection and then check the remaining cells
for the presence of the sFv by immunofluorescent staining for the
myc or HA epitopes.
Okay, that's all I have for now. I shall pass on any
other tips as they occur to me.
[No affiliation with Invitrogen, Dynal or The Simpsons]
Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD, USA)
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