OPEN LETTER to the Arabidopsis Research Community

Michael R. Sussman msussman at
Tue Aug 17 19:55:47 EST 1999

OPEN LETTER to the Arabidopsis Research Community

	We are in the process of establishing a user-fee service facility to
provide 'knockout' Arabidopsis mutant plants using T-DNA insertional
mutagenesis.  Seed money was provided from an NSF multiinstitutional
grant entitled "The Arabidopsis functional genomics initiative"
coordinated by Pamela Green at Michigan State University.  The basic
protocols are as described in Krysan, Young, Tax and Sussman,
<bold>Proc. Natl. Acad. Sci</bold>. 93:8145 (1996), but with a larger
population developed at the University of Wisconsin.  The new
population is comprised of 60,480 independently derived
kanamycin-resistant lines, developed in collaboration with the
laboratory of Rick Amasino in the Department of Biochemistry at
UW-Madison.  The population was created using vacuum infiltration and
<italic>Agrobacterium</italic>-mediated transformation.  For the
average 4,000 bp gene, this translates into a very high likelihood that
at least one knockout mutant of that gene exists in this population.
For genes smaller than the average, the likelihood of an insertion in
this population will be reduced, as predicted by the equation described
in Krysan et al (1996).  In the near future we anticipate the addition
of other deconvoluted large populations, as they become available.

	The 60,480 new lines are stored in 6,720 tubes each containing seed
from nine different T-DNA lines.  To screen this population for a
knockout in your gene of interest, we are using a PCR-based strategy
with gene specific primers.  In the near future, we plan to switch to
protocols that do not require gene specific primers, for amplification
of genomic sequence flanking the T-DNA.  Once perfected, these
procedures will allow us to create filters for hybridization screening
techniques as well as to determine the sequence of the plant DNA
flanking each insert.  When the Arabidopsis genome sequence is
completed, the database of flanking sequences in each line will provide
the ideal means of identifying and distributing knockout seeds in your
gene of interest.

	For the present and immediate future, our protocol is as follows. This
is also described in complete detail on the University of Wisconsin's
Biotechnology Center 'Knockout Plant Facility' website at

(1) You must synthesize two 29 bp long oligonucleotides, one at the 5'
end and one at the 3' end of your gene of interest.

(2) You will mail these primers to us and the facility will perform 32
PCR reactions with each of the two primers, together with a common
border primer.  The 64 PCR reactions will be mailed back to your lab,
where you will perform gel electrophoresis and a Southern blot.  From
the results of that blot you will know whether an insertion in your
gene of interest is present in the population.  The size of the PCR
band will tell you approximately where the insertion has landed in your
gene.  You should perform a sequencing reaction with that band and
these results will serve to confirm or refute the presence of a plant
containing at least one mutant allele, as well as to precisely
determine the exact location of the insert.  If the insertion allele is
of interest to your lab, the facility will perform 8 PCR reactions
using DNA from each of the pools of 225 lines, which comprise the
single 'super pool' of 1,800 identified in the first round.  The 8 PCR
reactions will be returned to your lab for another gel.  Once a single
pool of 225 has been identified, you will be sent seeds (ca. 200 seeds
per tube) from each of 25 pools of nine lines that you can use to
identify the individual knockout plant.

	To offset some costs that were not provided from grant funds, we are
charging users, $250 (plus shipping and handling) for the first round
of PCR reactions, and $250 for the next round, for a total of $500.
You may wish to budget these standard rates in future grant proposals.

<underline>General Comments

</underline>	This is a project of enormous magnitude, that is clearly
of timely importance to the Arabidopsis research community.  Our NSF
funding began on January 1, 1999 and we promised to have this facility
operational for screening 25-50 gene knockouts per week, by the end of
the first year (i.e., January 2000).  Since much of our effort is
devoted to producing seeds and DNA extractions of sufficient bulk for
the large anticipated demand, we may need to phase in the operation
gradually, so as not to overload the facility operation in its early
stages of existence.  Thus, depending on demand, we may need to limit
the number of requests per laboratory.  You may wish to prioritize your
requests and plan on having no more than five knockout searches active
at any one time.  Our timetable for operation in the first year is such
that we expect to be ready to accept requests on October 1, 1999 and
ordering will be accommodated automatically on our website.  The
following guidelines will be our rules of operation:

(1) You will be required to provide the ABRC at OSU with seeds of your
individual knockout plant, coincident with the first publication of
data involving the knockout.  Acknowledgement of the facility's efforts
in your publication would be appreciated and should reference the
original Krysan et al. (1996) report.

(2) There are no intellectual property restrictions on this service.
In other words, you are free to patent and license intellectual
property derived from the study of your individual mutant plant with
any private sector operations of your choice, without notification or
involvement of the University of Wisconsin.

(3) We will honor service requests from any active academic 'nonprofit'
laboratory.  Commercial 'for profit' laboratories should contact
Professor Rick Amasino or myself to discuss arrangements.

	I wish to emphasize that the gene specific primer PCR procedure is a
temporary measure to provide the best quality service while methods for
amplifying junction sequences without gene specific primers is
perfected, and while the genome sequencing project proceeds to

	The purpose of this communication is to provide you with a brief
description of the nature, cost and timetable for this service
facility.  If you have questions, please do not hesitate to contact the
facility manager Dr. Sandra Austin-Phillips (email address is
<underline><color><param>0000,0000,00FF</param>knockout at</color
 Also let me re-emphasize the large magnitude of this project, and
offer my request for your patience while we work out the bugs in its
early days of operation.


Michael R. Sussman

Professor and Director,

University of Wisconsin Biotechnology Center

425 Henry Mall

Madison, WI 53706


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