Hi:
I am very soon going to have introduce a couple of constructs into mouse
splenocytes (most likely activated by alloantigen). The goal is to test
if the gene we're interested in influences T lymphocyte lifespans and
"cloneability". The expression vectors will be introduced in the sense
and antisense orientations and then cloning frequency assessed. We've
electroporated a number of different cell types, including T cell clones,
but I've never attempted primary T cells. We have biorad gene pulser.
Does anybody out there have any experience with this type of thing, and
if so, could be please e-mail the electroporation parameters that you've
found to be the most effective? Thanks in advance.
While I'm here, I have a related question. While making stable
transfectants of CTL clones, I've been selecting in both G418 and
hygromycin. I already knew that CTL are notoriously resistant to G418,
but for some reason I had thought that their sensitivity to hyg. was a
bit more "normal". I've been finding that they are, in fact, quite
resistant to hyg also. I'm currently using 1.5 mg/ml G418 and 0.7 mg/ml
hyg. This ain't cheap! I'm starting to wonder if maybe the hygromycin I
just bought is a bit off. Anybody else out there have comments on
working concentrations of these drugs for selecting mouse CTL?
Eric
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Eric Atkinson * "When they said: 'Repent, repent',
Dept. of Biochemistry * I wondered what they meant".
University of Alberta * Leonard Cohen
Edmonton, Alberta *
Canada *
T6G 2H7 *
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