RE: [Histonet] IL1 KO mouse and cytokines in neurons

From:"Jacqui Detmar"

Hey there.  Yup, I agree that using KO mice as controls can be a
problem, although I work with about a dozen different KOs and have had
no problems, so I think it's a matter of knowing *what exactly* has been
knocked out, plus keeping your fingers crossed and praying to the right
gods .  Anyway, if you're worried about cytokine signaling to
adjacent cells (which was great thinking, Ray!), perhaps another
approach is to use either caspase-1 KO (mice available commercially) or
caspase-11 KO mice.  These enzymes process pro-IL-1alpha and beta to
their active, secreted forms.  Thus, the pro forms should be retained
within the primary cell types synthesizing the cytokines.  Both
caspase-11 and caspase-1 KO mice have been reported to exhibit very low
levels of secreted, processed IL-1beta and IL-1alpha.  I have not yet
seen anyone produce double caspase-1/caspase-11 KOs.  


Jacqui Detmar, Post-doctoral Fellow

Samuel Lunenfeld Research Institute, room 876

Mount Sinai Hospital

600 University Avenue

Toronto, ON, Canada

M5G 1X5


phone:   416-586-4800 x2451/x2290

fax:        416-586-8588




From: [] 
Sent: Monday, February 04, 2008 5:39 PM
To: Jacqui Detmar; Leyva-Grado, Victor;
Subject: RE: [Histonet] IL1 KO mouse and cytokines in neurons


Victor and Jacqui,


Could "some" neurons appearing to be immunoreactive to TNF alpha or Il-1
beta simply be the fact that they are cytokines?  Small molecular
weight, they are meant to be released from cells and not membrane bound
there or as part of the cell structure.  Glial cells, by the very fact
of what they are, are intimately associated with neurons.  How was
tissue fixed?  Maybe they are dispersed or leaking to a neighboring
neuron.  Many targets are anchored where they are, such as your NeuN
label, and that target is not going anywhere.  Cytokines do.  The
references of 10-15 years ago were novel then.  Not so now.  TNK K/o as
you said you can get.  At Wash State, you should have a good mouse
transgenics and mouse k/o husbandry facility.  With some Balb/c's, they
should be able to generate IL-1 alpha -/-. beta -/- or alpha/beta double
knock/outs.  Not that difficult for a good knock out genetics lab.  If
you are using the&n bsp;knock outs as control for the IHC staining (and
this applies to any use of knock-outs as negative controls for any IHC
staining) be sure you know EXACTLY what is knocked out. A mouse
frizzle/frazzle knock-out does not imply the total absence of mouse
frizzle/frazzel gene and protein.  One particular coding exon in a
multi-exon gene could leave the protein unresponsive or unusable in-vivo
or shortened but enough might be left, and if the proper epitope is
left, for IHC staining to occur.  Knock out is not the same as complete
absence.  So you can stain something that is "knocked out" and that is
why you need to be aware of EXACTLY what is knocked out.  Many people,
including yours truely, has been caught like this.




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