Hyg, Neo, and $$

Brian Foley brianf at med.uvm.edu
Thu Jun 16 19:06:02 EST 1994

down-n-out (eatkinso at gpu.srv.ualberta.ca) wrote:
: Here it is:  After you have selected for stable transfectants, how 
: important is it to keep the drug on the cells?

	That depends on what you are after.  It may have to be determined
empirically.  I too was frustrated by a lack of hard data on this sort
of thing in the literature. I scaned the library for weeks trying to find
out how many copies of plasmid per cell could be expected to integrate, how
many milligrams of protein per million cells might be produced, how stable
transfected lines are, and many other questions.  I found that for the 
most part, mammalian cell transfections are treated like a "black box".  
People just stick the DNA in there and if they get the result they want, 
then that is good enough.
	I did find interesting papers on "amplification" of inserted DNA, 
leading to greater expression of both the selectable marker gene, and 
other genes carried on the same plasmid.  Thus, if you keep the 
hygromycin on your cells, and even increase the dose over time, you may
get cells to produce more protein from your plasmid.  I am working
on this right now, because I just can't get enough of my protein of
interest.  It has also been claimed that you can increase the expression 
from co-transfected DNA through gene amplification.  The thought was that 
if the plasmids integrate at all, they often both integrate side-by-side
and thus amplification of one of them leads to amplification of the other.
I find this hard to beleive myself.

: I know that some people maintain all their drug-resistant cell lines in 
: drug-containing media.  As the person who was working with B cells said 
: though, this is *extremely* expensive when working with cells that have a 
: high baseline resistance.  I also know that many people remove the drug 
: ASAP, even before all the control cells are obviously dead, with the 
: argument that the drugs could be mutagenic and the less time the cells 
: are exposed to it, the better.

	Yes, the bottle of hygromycin I got from Calbiochem says it is 
suspected of being carcinogenic and a teratogen.  This would cause you
to beleive that it is also mutagenic.  The only thing I have done 
about this is to transfect a control lot of cells with the same plasmid
carrying no insert DNA.  These cells are maintained on hygromycin
right beside the cells transfected with the plasmid carrying my gene
of interest.  I have not yet seen any mutations in the system I screen for.

: I guess the reason that some people prefer to maintain the selective 
: pressure is to ensure that their inserted DNA is not kicked out of the 
: cell's genome, but how common is this really?  

	I think it has more to do with reversible amplification than
excision from the genome.  Gene amplification seems to be quite 
reversible over relatively short periods of time.
	The genetic markers most often cited for gene amplification
are HGPRT and GPT.  I don't have the references handy right now, but
write to me if you have trouble finding them.

: ES cells that are targeted 
: with a neo insertion are removed from drug ASAP and the neo cassette 
: remains stable.  When you think about the number of cell divisions 
: required to generate a mouse, that's a lot of generations.  This may be a 
: bad example, but I'm wondering if the frequency of foreign DNA 
: "rejection" has anything to do with the type of gene product.  It seems 
: reasonable to me that in the absence of drug selection, the introduced 
: DNA would be likely to be eliminated or selected against if it encoded 
: something that the cells were not entirely happy with.  

	I think that once it is integrated, it is pretty stable.  However,
if you have a mixed population from a mass transfection (rather than a cloned
and subcloned pure cel line from one transfected cell) you will have a
variety of cells, some producing more product than others.  In this mixed
population, some cells will outgrow others over time.  Thus even though
each cell line is stable, the population profile changes over time.

: If, however, the 
: foreign DNA is relatively benign, would this be more or less stable?
: From our own experience, we have been given a cell line that was 
: transfected with an expression vector encoding a cell surface marker.  
: The original selection was with G418, but we have never grown the cells 
: in the presence of drug, and through countless generations now, the 
: marker remains.  Obviously drug selection is not always necessary.

	Was it a clonal line?  Or a population of transfectants?
They still have G418 resistance, but is it he same level of resistance
that they once had?  My CHO-K1 wild-type cells die in 300 units/ml
of Hygromycin b in less than 3 days.  I have transfectants that
were selected in 300 U/ml then bumped up to 600 U/ml and then grown in
1000 U/ml.  I get better levels of expression of my gene now that they
are growing in 1000 U/ml than I did when they were first selected in 300
	The next step is to see how reversible this amplification is.

: I suppose that drug addition every now and then, just to weed out any 
: revertants would be a compromise between spending your whole grant on 
: G418 or hygromycin B and having all your cell lines end up back at the 
: wild type phenotype.
: What are other people's thoughts/experiences about this type of thing?  
: From the number of time's that I've been asked these very questions by 
: other people I'd say that there's a fair amount of confusion about this 
: out there.

	Yes, I would think that the whole field of molecular biology would
be better off if as much attention was paid to transfections as is paid 
to ligations and other DNA manipulations.  If I go to the library I can 
find all kinds of data about ligation efficiency and theory, but there
is very little on what happens to a plasmid after it is put into a
mammalian cell.  I'd like to see Southern blots or other data to indicate
how many copies per cell typically integrate.  I'd like to know why
the methods of transfection that give the best results in transient
transfections do not work well for stable transfections.  This does not
make sense to me...

: ********************************************************************
: Eric Atkinson	      *		"When they said: 'Repent, repent',
: Dept. of Biochemistry *		I wonder what they meant?"
: University of Alberta *			Leonard Cohen
: Edmonton, Alberta     *
: Canada		      *
: T6G 2H7		      *
: ********************************************************************

*  Brian Foley               *     If we knew what we were doing   *
*  Molecular Genetics Dept.  *     it wouldn't be called research  *
*  University of Vermont     *                                     *

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