T-DNA Generated Enhancer Traps in Arabidopsis

Overview of the project

How the lines are generated

Collecting seeds for distribution to the ABRC stock center

Detailed information about the lines

pD991 enhancer trap vector

Sample Patterns

Cloning flanking DNA

 

Overview of the project

This project involved the generation of more than 11,000 enhancer trap lines. Seeds for these lines have been sent to Arabidopsis Biological Resource Center at Ohio State University (ABRC) for distribution to the Arabidopsis community and presently pooled seeds are available from the ABRC and the Nottingham Arabidopsis Stock Center NASC. The ABRC is also distributing DNA pools from the T-DNA lines.

 

Detailed information about the lines

For all 12,000 lines we have and characterized the inflorescence staining pattern of the primary transformants. A summary of staining patterns that we have observed is available. Overall, 31% of the lines stain in the inflorescence. For a small number of lines we have information about staining patterns in the seedling. We have also compiled data generated and generously provided by other labs. The lab of Dr. Gary Drews at the University of Utah planted out seeds from about 1200 single lines and determined the kanamycin sensitive to kanamycin resistance ratio. The lab of Dr. John Bowman at UC Davis has looked in detail at the carpel staining patterns in the first 4000 lines.

 

The pD991 enhancer trap vector

The vector we used to generate the traps is called pD991. pD991 is a derivative of the binary plant transformation vector pCGN1547 (McBride and Summerfelt, Plant Mol. Biol. 14, 269-276 (1990)). The T-DNA of pD991 contains the -60CaMV minimal promoter fused to the uidA (GUS) gene. By itself the -60 CaMV promoter directs very low levels of transcription, and in the absence of a closely linked enhancer element, the level of GUS activity is undetectable. The -60 CaMV promoter sequences in the pD991 vector are located about 250 base pairs from the T-DNA right border sequence. After integration, enhancer elements in genomic DNA adjacent to the T-DNA insertion cause an increase in transcription from the -60 promoter resulting in an increased level of expression of the GUS gene, often in cell-, tissue-, or organ-specific expression patterns as directed by the adjacent enhancer. These patterns can be detected by staining for GUS activity using the chromogenic substrate X-gluc.

pD991 sequence adjacent to the right border

We have compiled about 2500 bp of sequence adjacent to the right border, from the SacI site through the 3' NOS trailer. The pD991 annotated sequence-right border contains the location of:

It is also possible to download a pD991 text file.

pD991 sequence adjacent to the left border

The pD991 sequence adjacent to the left border has also been determined.

 

How the lines were generated

We generated enhancer trap lines by vacuum infiltration transformation of Columbia gl1 plants. To identify transformants, we sowed these seeds collected from Agrobacterium-treated plants on plates containing kanamycin. In our hands, between 0.5-3.0% of the seeds from any given transformation are kanamycin resistant. We consider each kanamycin resistant seedling to be a separate enhancer trap line. To determine the inflorescence staining pattern, we cut off the primary inflorescence from the primary transformants and stain them in X-gluc. Details of our GUS staining protocol are available. Overall, about 30% of the lines exhibit a staining pattern in the inflorescence. In particular, we are interested in isolating lines that exhibit patterns during early stages of flower development. The details about how we sort through the traps are available in the form of a flow chart.

 

Collecting seeds for distribution through the ABRC stock center

For lines numbered 1-2600, we stained inflorescences from individual plants and collected seeds individually from these plants. For lines numbered 2600-4699, we stained individual plants and collected seeds from pools of 10 plants. For lines numbered higher than 4700, we stained inflorescences and collected seeds in pools of 10 plants.

If anyone has any questions about how these enhancer trap lines were generated or if they have any information about these lines they would like to make available on this web page, please contact Tom Jack (thomas.p.jack@dartmouth.edu).

 

Sample Patterns

 

Acknowledgements

This project was funded by a grant from the USDA (Plant Growth and Development). I would like to thank Peter McCourt, Gary Drews, Yuval Yeshed, and John Bowman for contributing information about the various enhancer trap lines.

Tom Jack
Department of Biological Sciences
Dartmouth College, Hanover, NH USA
Phone: (603) 646-3367
FAX: (603) 646-1347
 

More info about the Jack lab.

last updated 8/6/02

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