Arabidopsis Knockout Facility

[Sandra Austin-Phillips]

March 31, 2000

We are writing at this time with an update on the Arabidopsis 
knockout facility at the University of Wisconsin Biotechnology 
Center.  The facility opened in the fall of 1999, three months 
earlier than promised, and is now in full swing.  See the website 
www.biotech.wisc.edu/Arabidopsis for full details.  Approximately 200 
principal investigators from all over the world are currently using 
the facility  and many scores of valuable knockouts in specific genes 
of interest have already been obtained.  At present, the facility  is 
processing approximately 25-50 orders per week and as the need 
arises, we expect to increase throughput while keeping the cost and 
time required per screen to a minimum.  Of course, ultimately we hope 
to have all of the flanking DNA sequenced as a tag for each line, 
eliminating all of the wet work currently required.  Until that 
database is established, we are using the current procedure because 
it is the most reliable means of getting a knockout mutant !
!
into your hands.  There are approximately 27,000 predicted genes in 
the Arabidopsis genome, and the  population of  60,400 independent 
kanamycin resistant lines that we are currently using provides a 
high probability of obtaining a knockout in your average-sized gene 
of interest. This population is organized in 6,720 tubes of seed, 
each derived from nine different kanamycin resistant lines.  We 
recently finished bulking this population and a large amount of seed 
has been deposited at the Ohio State University Arabidopsis Seed 
Stock Center.  Additional populations are being developed and will be 
incorporated into the facility as quickly as our resources  allow.

Early on in this project in our own labs we  noted that a very small 
percentage (ca. 10%) of the  60,400 line population exhibited some 
fertility problems. However, we decided to proceed with the use of 
this population as knockouts because the number of lines that showed 
a problem were so few. The infertility  may be related to the 
presence of a couple of  hundred base pairs of the AP3 promoter in 
the  T-DNA construct used for mutagenesis.  Regardless of the cause 
and even though the percentage is small,  if your knockout plant 
shows such sterility problems, we will quickly, and at no added 
expense, scan a new population for your gene of interest.  This new 
population contains ca. 70,000 lines and although it is not yet ready 
for general use (e.g. seeds have not been bulked  and DNA preps are 
still in preparation ) we will provide its use, free of charge, for 
the few investigators with lines showing sterility problems.  The new 
population has absolutely no Arabidopsis sequence!
!
  in its vector and thus, if the sterility problem persists in your 
new knockout line, an alternative explanation, such as a requirement 
of your gene for fertility,  needs to be invoked.

Some final comments.  As we explained in our recent review ("T-DNA as 
an insertional mutagen in Arabidopsis", Plant Cell 11:2283-2290), the 
isolation of one knockout allele for your gene of interest is the 
beginning of a potentially long and fruitful road of discovery for 
its in planta function.  However, don't underestimate the work 
required to conclusively establish a causal link between an insertion 
and observed phenotypic changes (cf. Figure 4).  Knockouts are 
obviously a very powerful approach for establishing gene function, 
and we will do our best to get these plants into your hands at the 
very earliest time.

Sincerely,



Michael R. Sussman
Professor of Genetics and Horticulture
and Director of the Biotechnology Center


Richard Amasino
Professor of Biochemistry




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