Session 2 GENE KNOCKOUT SYSTEMS USING Ac/Ds


The maize two component Ac/Ds transposable element system has been shown to function effectively within rice cells and represents a powerful tool for tractable gene mutagenesis. This approach complements T-DNA tagging strategies discussed in session 1 for saturation tagging of the rice genome. Combined these two approaches will facilitate the ambitious task of generating 500, 000 independently generated and characterized rice insertion lines.

All speakers in this session reported exceptional progress in generating Ds tagged rice lines with tens of thousands of such lines already generated and partially characterized. Sophisticated Ac/Ds constructs are being utilized which improve transposition and screening efficiencies by employing excision markers, insertion markers and counter selection strategies. DNA sequences flanking Ds insertion sites are being obtained by PCR and localized in the rice genome by either conventional gene mapping or by homology to the rice genome sequence. Databases of such sequences are being constructed and bioinformatics analyses undertaken. Tagged genes have been identified based upon phenotypic analysis, expression analysis using gene trap and reporter trap systems and DNA sequence homology.

Ds elements, while preferentially moving to linked locations, have none the less been shown to distribute in a relatively random fashion throughout the genome, although an apparent preference for chromosome 1 was reported. Facilitating the use of these elements as mutagens is the reported preferential insertion of these sequences into gene rich regions and gene coding regions.

Efforts to further increase the efficiency of obtaining and identifying Ds tagged rice lines were discussed by all speakers. Strategies to improve this transposition and screening process included;
1) Increasing Ds transposition in germ cells using meiosis-associated promoters to express
transposase.
2) More efficient selection and counter-selection strategies.
3) Hybridising cDNA to flanking Ds sequences to identify tagged genes expressed in a
tissue of interest.
4) Using rice lines containing multiple Ds elements and that show high transposition
frequencies in subsequent generations.
5) Re-activating Ds transposition by regenerating plants in tissue culture.
6) Transient expression of transposase.

The substantial progress made in generating Ds tagged lines makes the task of saturating the rice genome appear feasible. However, potentially compounding this process is the identification of mutant phenotypes not associated with insertion sequences. Multiple insertions per gene are therefore highly desirable. Given the magnitude of the task, the formation of an international consortium to achieve the goal of a functional ‘rice gene machine’ appears to be both an essential and logical step for success.

Reporter: M. Ayliffe