Geneticin/Kanamycin; interchangeable?
Alex Brands
I was planning to use kanamycin resistance as a selectable marker in
yeast, and I aquired a construct from another lab that has a kanamycin
resistance cassette. However, my negative control plates revealed that
the parent yeast (w303) was not sensitive to the kanamycin. After talking
to the other lab, I found out that they use something called Geneticin,
(that name is a registered trademark of GIBCO) which is about 20 times
more expensive than kanamycin.
The formula for geneticin is C(20)H(40)N(4)O(10)*2H2SO4
The formula for kanamycin is C(18)H(36)N(4)O(11)*1H2SO4
So, they are different, but not by much. Is geneticin just some modified
version of kanamycin?
The GIBCO catalog entry for geneticin says that it may be stored in powder
form for 1 year, and the bottle of kanamycin I used was definitely much
older than that. However, everyone I've asked says that kanamycin powder
is good almost indefinitely in the fridge.
So, is my kanamycin simply expired, or are yeast not sensitive to
kanamycin? Am I stuck with Geneticin?
All help is appreciated very much!
Alex Brands
Washington University
David L. Haviland, Ph.D.
Alex:
Ohhhh!! Good one... Kan and Geneticin are both aminoglycosides and act, if
memory serves, the same way. However, kan is specific for *prokaryotic*
ribosomes and geneticin (neomycin) is specific for *eukaryotic* ribosomes.
You have you use what you need depending upon the host you are using as
they can not be intermixed.
For example, Stratagene makes a nice vector called pBK-CMV which has NeoR
as its resistance marker. It is a nice vector in that for bacteria, one
uses kanamycin and gets no satilite colonies. The advantage is then had in
that you can take the vector, plunk it into your cell of choice, and use
G418 as your eukaryotic selection marker.
For your case, it is the same thing: you use kan while it is in bacteria,
but for your application in yeast, sorry... you're stuck and do need G418
(geneticin). BTW- G418 does have inter-lot variability and ideally should
be tested in a killing curve with each new lot purchased....
Hope this helps.
Former WU-er...
David
=============================
David L. Haviland, Ph.D.
Asst. Prof. Immunology
University of Texas - Houston, H.S.C.
Institute of Molecular Medicine
2121 W. Holcombe Blvd.
Houston, TX 77030
Internet:"dhav...@imm2.imm.uth.tmc.edu"
Voice: 713.500.2413 FAX: 713.500.2424
------------------------------------------------------
Never take life seriously. Nobody gets out alive anyway.
=============================
Mikhail Alexeyev
At 12:18 7/11/97 -0500, Alex Brands wrote:
>
>Ohhhh!! Good one... Kan and Geneticin are both aminoglycosides and act, >if
>memory serves, the same way. However, kan is specific for *prokaryotic*
>ribosomes and geneticin (neomycin) is specific for *eukaryotic* >ribosomes.
If I am not mistaken, resistance to kanamycin is being used as selective
marker in genetic transformation of plants, which are eucaryotes.
However, eucaryotic ribosomes are less sensitive to kanamycin. On the
other hand, according to the patent claims that Gibco refers to,
Geneticin (G418 sulfate) is toxic to bacteria.
I have thought that the whole isssue was in selective permeability of
membranes to antibiotics in addition to (likely) potential differences
in ribosomal inhibition. For example, gentamicin, ancestor (?) of G418
cannot penetrate mammalian cells in culture and oftentimes is being used
in experiments that are aimed at determination of bacterial invasion of
cultured cells: gentamycin kills bacteria outside but not inside
eucariotic host.
Same is likely to be the case with yeast.
M.A.