The Arabidopsis Knockout Facility at the University of Wisconsin–Madison

Abstract

As of this writing the Arabidopsis genome is 97% sequenced with only small portions of the highly repetitive regions within centromeres and telomeres remaining. The identification of approximately 25,000 plant genes will give plant biologists an opportunity to identify and understand the function of the proteins they encode. One exciting tool that will aid in this endeavor is the use of insertional mutagenesis to create "gene knockouts." The availability of a mutant line in which the action of a known, specific gene has been disrupted gives the plant biologist a powerful tool in understanding the action of that gene. The basis of this approach is to create a large population of plants containing randomly inserted pieces of foreign DNA. If the sequence of a gene is known, it is possible to devise a PCR-based strategy to identify a plant where that specific gene has been disrupted by the insertion of foreign DNA. To fully utilize this technology it is necessary to saturate the genome with insertion mutations and to develop efficient PCRbased screening methods to comb through knockout plant populations and identify specific mutant plants. The smaller the gene, the more difficult a target it represents, and thus hundreds of thousands of lines are needed to provide a high probability that a particular gene is present as a knockout in the population.As a beginning, a method has been developed for rapidly searching a large collection of T-DNA transformed Arabidopsis lines for the presence of T-DNA inserts within specific genes (Krysan et al., 1996(Krysan et al., , 1999. To use this technology, a collection of 60,480 Arabidopsis (accession Wassilewskija [WS]) lines were generated that were transformed with the T-DNA vector pD991. Preliminary data from screening this collection indicated that this collection could indeed be efficiently screened for mutant lines. To share this resource with all members of the Arabidopsis research community, a "Knockout Facility" was established at the University of Wisconsin (Madison) in 1999 as part of the Arabidopsis Functional Genomics Consortium. A detailed description of this initial population and the operation of the facility have been given in a recent publication (Krysan et al., 1999) and is described more fully at the website http://www. biotech.wisc.edu/Arabidopsis.In this brief note we will give an overview of the first year's operation and describe our future plans. We strongly advice that users read the web site fully before using the facility and contact us with any questions that they may have. CURRENT FACILITY OPERATIONThe facility is housed in the Plant Biotechnology Laboratory at the University of Wisconsin Biotechnology Center located at 425 Henry Mall, University of Wisconsin, Madison, WI 53706. The fee-for-service operation relies heavily on the administrative and information services of the Biotechnology Center. Users interact with the facility through the website. The site has a full description of the PCR screening including primer design and subsequent analysi...

Repression of theLEAFY COTYLEDON 1/B3Regulatory Network in Plant Embryo Development byVP1/ABSCISIC ACID INSENSITIVE 3-LIKEB3 Genes

“…We obtained T-DNA insertion alleles, val1-1, val2-1, val2-2, and val3-3 in Ws ecotype from the University of Wisconsin Arabidopsis Knockout facility (Sussman et al, 2000). The val3-3 allele, which has a T-DNA insertion within a coding exon of the VAL3 gene, was derived from an activation tag population.…”

Double Knockouts of Phospholipases Dζ1 and Dζ2 in Arabidopsis Affect Root Elongation during Phosphate-Limited Growth But Do Not Affect Root Hair Patterning

“…Homozygous mutant plants were isolated using the T-DNA left-border primer and genespecific primers: Primers 1a and 1b were used to identify pldz1 and primers 2a and 2b to identify pldz2 (Table I). pldz1-ws and pldz2-ws insertion mutants are in the Ws background, and mutants were identified by screening pools of T-DNA insertion lines according to the protocol of the Arabidopsis Gene Knockout Research Facility at the University of Wisconsin (Sussman et al, 2000). PCR was carried out on pooled genomic DNA with a T-DNA left-border primer, JL202, and gene-specific primers, 1e and 1f for the PLDz1 gene and 2e and 2f for the PLDz2 gene.…”

Protein Geranylgeranyltransferase I Is Involved in Specific Aspects of Abscisic Acid and Auxin Signaling in Arabidopsis

“…Plant seed stocks were obtained from the following suppliers: ggb-1 was identified by PCR screening the T-DNA knockout collection at the University of Wisconsin Arabidopsis Knockout Facility (Krysan et al, 1999;Sussman et al, 2000) and obtained along with the corresponding wild-type line (Ws); ggb-2 was identified by the Salk Institute Genomic Analysis Laboratory by thermal asymmetric interlaced PCR and obtained from the Arabidopsis Biological Resource Center along with the corresponding wild-type line (Col). ggb-1 and ggb-2 were backcrossed to Ws and Col, respectively, and recovered as homozygotes in the F 2 generation.…”

“…As shown in Figure 1, A and B, GGB consists of 11 exons and 10 introns. Two distinct T-DNA insertion mutations were identified in the GGB coding region: one from the University of Wisconsin Arabidopsis Knockout Facility (Krysan et al, 1999;Sussman et al, 2000) in the Wassilewskija (Ws) background called ggb-1 and one from the Salk Institute Genomic Analysis Laboratory in the Columbia (Col) background called ggb-2. In ggb-1, the T-DNA is located in intron 10, whereas in ggb-2, the T-DNA is located in exon 1.…”