mSplenocyte Electroporation

down-n-out eatkinso at gpu.srv.ualberta.ca
Fri Jun 3 12:22:28 EST 1994

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 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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