Hygromycin info.

Nobody nobody at hgmp.mrc.ac.uk
Thu Feb 28 14:31:25 EST 2002


Hi, Everyone,
I post the question about TAC transformation and hygromycin
selection a while ago. Here is a collection of answers and
information I=92ve found out of varies sources. I haven=92t been
lucky to get it work for me. If anyone has positive results, please
send me your protocols. Your help will be highly appreciated.
=46rom: Randall Kerstetter { HYPERLINK "mailto:randallk at sas.upenn.edu"=20
}randallk at sas.upenn.edu
Typically, I perform Hygromycin selection at 30mg/L .
I sow sterilized seeds on plates consisting of
=20  1/2x MS salts -- pH adjusted to 5.7 with KOH
=20  0.8% bactoagar
=20  no sucrose
=20  adding hygromycin after autoclaving.
Avoid contact or breathing hygromycin vapor when pouring
plates -- it is
pretty toxic to humans.

Seeds are stratified at 4 degrees for 3 days, then moved to a
dark place
at room temperature for 3 to 5 days (a drawer is good).  The
Hyg resistant
seedlings are those that show good hypocotyl elongation in the
dark.
Transfer the plates to the light when you can score hypocotyl
elongation.
Transplant green seedlings with long hypocotyls to soil.  You
don't want
the seedlings to touch the lid of the plate, especially if there is
condensation on it.  Avoid seedlings with long hypocotyls that
arise from
seeds that were not in contact with the agar.
In 1997 archive,
=46rom: { HYPERLINK=20
"mailto:dfzhou at vt.edu&replyto=3Dkrs1-3001971517500001 at nadgee.mrc-lmb.cam=20
=2Eac.uk&subject=3DRe:%20Hygromycin%20concentration" }dfzhou at vt.edu=20
(Dafeng Zhou)
Hi, I have post the same question some times ago. Here is my
impression of =0F
the answers: the uppper limit of HYG is 30 microgram/ml, some
people even =0F
use as little as 15 ug/ml. Right now, I am using 30 ug/ml, looks
pretty =0F
good. The difference between Kanamycin and HYG is the
kanamycin selection is =0F
easy to seeb(non transformed plants bleached in about 2 week).
However, the =0F
HYG selection does have this advantage, the selection needs a
little longer =0F
(3 weeks) before you can tell the difference based on =0F
selection(nontransformed no longer grow and no real leaves)
=46rom: Dave Twell <{ HYPERLINK=20
"mailto:twe at leicester.ac.uk&replyto=3Dkrs1-3001971517500001 at nadgee.mrc-l=20
mb.cam.ac.uk&subject=3DRe:%20Hygromycin%20concentration"=20
}twe at leicester.ac.uk>
I don't know your vector but we are using hyg selection in C24 =0F
transformants and find that 5 =0F
mg/l usually works if you are looking for a minimum, 10 is better
and 15 =0F
still better but may =0F
start to have an effect on resistant plants.
=46rom: Nicole Bechtold <{ HYPERLINK=20
"mailto:bechtold at oxygene.versailles.inra.fr&replyto=3Dkrs1-3001971517500=20
001 at nadgee.mrc-lmb.cam.ac.uk&subject=3DRe:%20Hygromycin%20concentration"=20
}bechtold at oxygene.versailles.inra.fr>
I use 25 mg/l hygromycine and the selection is very clear and
rapid. =0F
The frequency of transformation is variable, some times you can
select 5 =0F
transformants per plate of 300-500 seeds some times 1 or 0.
=46rom: Grazyna Rempulska <{ HYPERLINK=20
"mailto:gbujas at botinst.unizh.ch&replyto=3Dkrs1-3001971517500001 at nadgee.m=20
rc-lmb.cam.ac.uk&subject=3DRe:%20Hygromycin%20concentration"=20
}gbujas at botinst.unizh.ch>
Usually I used the concentration of 20 mg of hygromycin B per
L =0F
medium for the selection of A. thaliana transformants after
infiltration. =0F
I performed my transformation with pGDW 3.1 vector (from
Dr. C. Koncz) and =0F
pGV3101 strain of A. tumefaciens. You have to wait few weeks =0F
(3-4 weeks) after sowing and your selection becomes clear. On
the begin =0F
all plants will be green or light green, and after few weeks
(seedlings in =0F
the stage of two cotyledones and two well developed leaves) the
selection =0F
will be obvious: only transformed plants will survive on hygr.
containing =0F
medium and remain green.
Articles about TAC transformation at Pubmed,

_1: =13
{ HYPERLINK=20
"http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=3DRetrieve&db=3DPubM=
=20
ed&list_uids=3D11814697&dopt=3DAbstract" }Liu YG, Liu H, Chen L, Qiu W,=20
Zhang Q, Wu H, Yang C, Su J, Wang Z,
Tian D, Mei M.=13
{ HYPERLINK=20
"http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=3DPubMed&cmd=3DDispla=
=20
y&dopt=3Dpubmed_pubmed&from_uid=3D11814697" }Related Articles=13
=12
=20    Development of new transformation-competent artificial
=20    chromosome vectors and rice genomic libraries for
=20    efficient gene cloning.=0F
=20    Gene. 2002 Jan 9;282(1-2):247-255.=0F
=20    PMID: 11814697 [PubMed - as supplied by publisher]
_2: =13
{ HYPERLINK=20
"http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?cmd=3DRetrieve&db=3DPubM=
=20
ed&list_uids=3D10339623&dopt=3DAbstract" }Liu YG, Shirano Y, Fukaki H,=20
Yanai Y, Tasaka M, Tabata S, Shibata D.=13
{ HYPERLINK=20
"http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=3DPubMed&cmd=3DDispla=
=20
y&dopt=3Dpubmed_pubmed&from_uid=3D10339623" }Related Articles, {=20
HYPERLINK=20
"http://www.ncbi.nlm.nih.gov:80/entrez/query.fcgi?db=3DPubMed&cmd=3DDispla=
=20
y&dopt=3Dpubmed_nucleotide&from_uid=3D10339623" }Nucleotide,
{ HYPERLINK=20
"http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=3DRetrieve&db=3DPubMed&=
=20
dopt=3DAbstract&list_uids=3D10339623" }=0EFree in PMC=0E =13
=12
=20    Complementation of plant mutants with large genomic
=20    DNA fragments by a transformation-competent artificial
=20    chromosome vector accelerates positional cloning.=0F
=20    Proc Natl Acad Sci U S A. 1999 May 25;96(11):6535-40.=0F
=20    PMID: 10339623 [PubMed - indexed for MEDLINE]
Hygromycin information from web search,
Description: Hygromycin B, an aminoglycosidic antibiotic produced
by Streptomyces hygroscopicus, is used for the selection and
maintenance of prokaryotic and eukaryotic cells transfected with the
hygromycin resistance gene, hphr. Hygromycin B kills bacteria, fungi
and higher eukaryotic cells by inhibiting protein synthesis. The
resistance gene codes for a kinase that inactivates Hygromycin B
through phosphorylation. Cloning of the resistance gene and fusion
with eukaryotic promoters has resulted in the development of vectors
that permit selection for resistance to Hygromycin B in both
prokaryotic and eukaryotic cells.
Use:  The working concentration for the purpose of selection varies with cel=
l
type, media, growth conditions and cell metabolic rate. Recommended
concentration for the selection of resistant cells is 25-1000 mg/ml. Commonl=
y
used concentrations for selection are 200 mg/ml for mammalian cells, 20-200
mg/ml for plant cells & bacteria cells and 200-100 mg/ml for fungi.=20
Your optimum
concentration should be tested experimentally
STABILITY AND STORAGE :
Hygromycin B solution is stable for at least one year at 4=B0C and
at least one month at 37=B0C. It is shipped at room temperature.
Hygromycin B is sensitive to high concentrations of acid but a
short-term exposure to dilute acids can be tolerated.
CONDITIONS OF SELECTION :
Most cells growing aerobically are killed by Hygromycin B in the
concentration range of 10 to 1000 =B5g/ml. However, the
sensitivity of cells is pH dependent, i.e., the higher the pH of
culture medium, the greater the sensitivity. Thus, the
concentration of Hygromycin B required for complete growth
inhibition of given cells can be reduced by increasing the pH of
the medium. In addition, using low salt media whenever possible
decreases the amount of Hygromycin B needed.
Thanks for all helps,
Li

---




More information about the Arab-gen mailing list