PC12 Stable transfectant

Min-Ho Lee mhlee at WWW.BRIC.POSTECH.AC.KR
Sun Dec 29 19:23:39 EST 1996


Lee, Hyoung-gon wrote:
> 
> Hi!
> I want to know about efficient transfection method for stable
> transfection on PC12 cell; Good vector, good protocols,....
> Anyone who know about this method, Please tell me about.
> 
> Hyoung-gon, Lee

Hello!
I've collected about bio-methods news. I couldn't find the transfection
method on PC12 cell, but I find transfection methods on other cell lines
as follows.

Min-Ho Lee : mhlee at bric.postech.ac.kr
Web for the Korean Entomologist (my home page):
http://bric.postech.ac.kr/about/mhlee/
BRIC (Biological Research Information Center), POSTECH, Korea
http://bric.postech.ac.kr

-------------------------------------
Subject: Re: Wanted: Method for transfection of primary human
fibroblasts
Date: Thu, 21 Nov 1996 | From: bernard at elsie.nci.nih.gov (Bernard
Murray)

Hi All, I am having toxicity problems with lipofectamine in transfection
assays on primary human
fibroblasts. Can anyone suggest a protocol that results in reasonable
transfection efficiency of primary
human fibroblasts? Even with optimization I am only getting 2-3 cells in
a field of 10E7 cells by direct
immunofluorescence. 

I've no idea how stringent your optimisation was so I can only offer
suggestions along these
lines.

You have to optimise *all* the variables to get the best transfection
efficiency. There's no such
thing as a protocol that is good for all cell types. Plate cells in eg.
6 well dishes and try a range
of;

a) Lipofectamine concentrations (my rough test is 2.5, 5.5 and 22.5
ul/well)
b) DNA concentrations (eg. 1, 3, 10 or more ug/well)
NOTE: You have to vary both of the above as both the total amounts and
the ratio of the two
components is important.
c) Initial cell density (eg. 30 - 80% confluence - the faster they grow
the lower the starting
density)
d) Exposure time (the longer the better - some cells can only stand a
few hours before there are
signs of toxicity, others can be exposed overnight).

I usually use double-banded pCMVbeta as the "gold standard" and assess
the transfection
efficiency by staining in situ with X-gal. Otherwise I use the general
protocol that Gibco/BRL
supplies and expose the cells to the complexes in OptiMEM in the absence
of serum then
remove and feed with normal medium. Some cells just don't like to be
transfected and you have
to push them to the border of significant toxicity (so a high percentage
of the remaining viable
cells are positive). You also use cell in good condition and that are
growing exponentially before
they are plated. From what I understand, fibroblast lines (eg. 3T3
cells) can be transfected with
high efficiency so you should be able to find a starting protocol
somewhere. A few days of work
spent in optimisation is well worth the effort.

If you've tried all this then I guess its time to try a different
method. Some colleagues found
switching from Lipofectamine to Lipofectin was a help for some cell
types.
Good luck,

Bernard

Bernard Murray, Ph.D.
bernard at elsie.nci.nih.gov (National Cancer Institute, NIH, Bethesda MD,
USA) 
-----------------------------------------------------
Subject: Re: 293 Cells: how to transfect? 

Date: Sun, 23 Jun 1996 19:42:44 GMT From: amcshea at world.std.com (Andy) |
To: methods at net.bio.net 

iddavis at vms.cis.pitt.edu wrote:

jstrassw at opal.tufts.edu writes: Hello! I am trying to use 293 cells
because they are reported to be
highly transfectable (>50% by Calcium Phosphate) and they are
EGF-responsive. The only problem is
that the buggers dont adhere well to dishes. I am loosing a lot of cells
every time I wash after
transfection. Any suggestions? 

Stick them down on collagen/poly lysine, dont leave them long at room
temp, try and avoid
washing them in PBS with not a lot else in it. I use lipofectamine at 5
microlitres to 1 microgram
DNA and tranfect in the presence of 0.5% serum in DMEM overnight and get
good efficiency
(70%+). They are so transfectable its worth the lack of stickiness.
Alternatively select for a
sticker sub-clone of 293's (depends what you are trying to do). Good
luck, 
----------------------------------------------

Subject: Re: 293 Cells: how to transfect? 

Date: 21 Jun 96 15:00:10 EDT From: iddavis at vms.cis.pitt.edu | To:
methods at net.bio.net 

jstrassw at opal.tufts.edu writes: Hello! I am trying to use 293 cells
because they are reported to be
highly transfectable (>50% by Calcium Phosphate) and they are
EGF-responsive. The only problem is
that the buggers dont adhere well to dishes. I am loosing a lot of cells
every time I wash after
transfection. Any suggestions? 

Try using a milder detergent and don't use the scourer quite so much.
Also, 293 cells are
notorious for not being suitable for dishwashers - perhaps this is where
your problem lies. 

Seriously though, this shouldn't be a problem: centrifuge the pellet and
make sure you throw the
right bit away! 

Ian Davis iddavis at vms.cis.pitt.edu 
-----------------------------------------------
Subject: Re: Transfection of HeLa cells
Date: Wed, 11 Sep 1996 | From: jstrassw at OPAL.TUFTS.EDU

Agustin Alconada wrote: Hello everyone, Does anybody know how the
different promoters found in
common eukaryotic expression vectors compare in terms of expression
levels in HeLa cells? For
example, should I expect higher expression levels of my protein if I
transfect the HeLa cells with a
SV40, a SFFV- or a CMV-promoter based vector? Thanks


Hello! I can not say which is better because other factors (splice
sasette in plasmid, 5' and 5'
ujntranslated sequences) definately affect expression. We use CMV-based
plasmids for
expression in HeLa, but our transfection comtrol plasmid is SV40
promoter/enhancer B-gal
construct. I would like to know the answer to your question too.

Good Luck,
John
John Strasswimmer, MD,PhD Candidate Tufts - New England Medical Center
Box 166 Boston,
MA 02111 USA
Phone (617) 636 8396 / Fax (617) 636 6190
Email: jstrassw at opal.tufts.edu
HTTP://WWW.healthsci.tufts.edu/microbiology/strass/JSpage.HTML
----------------------------------------

Subject: Re: Transfection of HeLa cells
Date: Wed, 11 Sep 1996 | From: rkoedood at bio.bu.edu (Riekeltje Koedood)

I recently compared MSV, CMV and SV40 promoter/enhancers driving lacZ
genes in HeLa
cells. The CMV plasmid won by far (about 40-100 times higher than SV,
whereas MSV didn't
seem to work at all). Of course other things also play a role (as john
mentioned), and I don't
know much else about these vectors (was just looking for a good
reference gene).

Marieke Koedood
Boston University
-------------------------------------------



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