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[Octopush1]

Hi All,

I'm new to MEFs and cell culture in general. I normally go only so far as to make a few primary cell cultures from embryos when and where I need them (and normally chicken not mice). But I did have some mice embryonic fibroblasts in culture from a C57BL6/CD1 background (both wt and knockout MEFs). They went senescent...and then the knockout MEFs started growing again. I guess they've become immortalised? I've expanded them and now have put them through 5 passages up to 100cm2 flasks, so I can freeze them down. They are from a very useful mutant line, which are usually early embryo lethal. So...great this is a very nice thing to happen. But I've got no controls! The control wt culture never became immortalised, so what do I do now? Can I use something like a 3T3 line as a control? Make primary fibroblasts? I'd like to do a microarray asap, so I can compare gene expression to my other mutants. Any ideas? Also does this mean my gene of interest, which was knocked-out could be a tumor-supressor?
Thanks Octopush

[bob1]

Spontaneous immortalization does occur occasionally in primary lines, though it is quite rare, unless you happen to alter a few key pathways: ko of p53, pRB and activation of a telomere lengthing mechanism.

You won't have an isotope control, from your wildtypes, but you should be able to use well characterized cells such as the 3t3 line, or perhaps you could try immortalzing your own wild types by transfecting in SV-40 large T antigen.