Scientist Solutions - LacZ fixation

cellbio45 Posted 8/20/2009 12:14:37 PM	I was having little trouble with a lacZ tissue staining. I am working with 'knock in mice' and wanted to ensure that the gene was present in the tissue. So the said plasmid carried a lacZ gene. upon harvesting, our team over fixed the tissue and we received no blue coloration in our tissue, were we are almost sure that the plasmid was taken up. I have heard of some antigen retrieval processes used in histochemistry, but does anyone know if there is a process to "fix" the over fixation in this case and retrieve the B-galactose, so that I will not have to redo my experiment? Any suggestions would be helpful.
parvoman Posted 8/20/2009 4:01:04 PM	Are these transgenic mice? ie. Should all cells have at least one copy of the plasmid? Or have you administered plasmid to a normal mouse? Which tissue(s) do you expect to have beta-galactosidase expression in? Have you tried doing PCR to detect part of the introduced plasmid?
cellbio45 Posted 8/20/2009 6:24:10 PM	These were transgenic mice, but we only bread one for the this specific mutation. The PCR showed presence of the gene. Were were testing the lung tissue and had fixed, frozen and taken cross sections of the entire lung.
parvoman Posted 8/21/2009 2:20:41 AM	I had a look back at a paper puplished by a guy I used to work with. He was looking at beta-gal expression in mouse livers and did whole organ fixation and staining. Pattern and load of spontaneous liver metastasis dependent on host immune status studied with a lacZ transduced lymphoma. Krüger A, Umansky V, Rocha M, Hacker HJ, Schirrmacher V, von Hoegen P. Blood. 1994 Nov 1;84(9):3166-74. The methods section describes how the whole organ staining was carried out. Did I understand correctly that this is the only animal you have, ie. there is no TG colony?
cellbio45 Posted 8/21/2009 10:42:13 AM	Thank you for all of your help. This was the only viable mouse that we obtained for this experiment, but after speaking with a few different sources. Think we are going to have to start over. Again your attention has been much appreciated.
dacrotty Posted 9/29/2009 2:57:12 PM	Protocol for staining mouse embryos for B-galactosidase (lacZ) available here: http://cshprotocols.cshlp.org/cgi/content/full/2007/8/pdb.prot4725
pearlpraba Posted 9/29/2009 3:04:38 PM	praise the lord Transcription of the genes that code for proteins involved innitrogen fixation in free-living diazotrophs is typically repressedby high internal oxygen concentrations or exogenous fixed nitrogen.The DNA sequence of a regulatory locus required for repressionof Rhodobacter capsulatus nitrogen fixation genes was determined.It was shown that this locus, defined by Tn5 insertions andby ethyl methanesulfonate-derived mutations, is homologous tothe glnB gene of other organisms. The R. capsulatus glnB genewas upstream of glnA, the gene for glutamine synthetase, ina glnBA operon. beta-Galactosidase expression from an R. capsulatusglnBA-lacZ translational fusion was increased twofold in cellsinduced by nitrogen limitation relative to that in cells undernitrogen-sufficient conditions. R. capsulatus nifR1, a genethat was previously shown to be homologous to ntrC and thatis required for transcription of nitrogen fixation genes, wasresponsible for approximately 50% of the transcriptional activationof this glnBA fusion in cells induced under nitrogen-limitingconditions. R. capsulatus GLNB, NIFR1, and NIFR2 (a proteinhomologous to NTRB) were proposed to transduce the nitrogenstatus in the cell into repression or activation of other R.capsulatus nif genes. Repression of nif genes in response tooxygen was still present in R. capsulatus glnB mutants and musthave occurred at a different level of control in the regulatorycircuit.