The IMA Ds Insertion lines were generated by Prof. Sundaresan and colleagues from the Institute of Molecular Agrobiology (IMA) at the National University of Singapore.

This system utilises a modified maize Dissociation (Ds) transposable element carrying a ß-glucuronidase (GUS) reported gene that acts as either a gene trap or an enhancer trap for the detection of genes by their expression pattern. Specifically, various starter lines, each containing a single stable Ds insertion, were crossed with lines expressing the Activator (Ac) element transposase so as to initiate transposition of the Ds element.

Selectable and Counterselectable Markers

The Ds element carries the NPTII gene, which confers resistance to kanamycin, and a modified GUS reporter gene. Thus, plants representing a transposition event can be selected by virtue of the kanamycin resistance gene that is contained within the Ds element.

The parental Ac transposase gene is been linked to the indole acetic acid hydrolase (IAAH) gene, which confers sensitivity to naphthalene acetamide. Progeny retaining the T-DNA donor site are eliminated, thus, progeny can be selected that are free of transposase activity. See Figure 1 below.

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Figure 1: Schematic Diagram of the Ds Donor Site and Possible Transposition Events. Open arrowheads indicate the 5' and 3' ends of the transposon. The Ds element carries the NPTII gene, which confers resistance to kanamycin, and a modified GUS reporter gene ( Sundaresan et al. 1995). Possible transposition events include the following: (1) unlinked or loosely linked transposition to the same chromosome; (2) transposition to a different chromosome; (3) closely linked transposition; and (4) closely linked transposition disrupting the IAAH gene. Hatched boxes represent left and right T-DNA borders (T-DNA-LB and T-DNA-RB, respectively).

In this way, Ds elements that remain closely linked to the T-DNA donor site by virtue of a proximal transposition event can be excluded, and only transposition to more distal sites in the genome results in plants that will survive the selection regime.

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PCR Amplification

The following nested primers, complementary to the 3’ and 5’ ends of the Ds element, are used:

Complementary to the 3’ end:

Ds3'-1a (GGT TCC CGT CCG ATT TCG ACT)

Ds3'-2a (CGA TTA CCG TAT TTA TCC CGT TC)

Ds3'-3a (TCG TTT CCG TCC CGC AAG T)

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Complementary to the 5’ end:

Ds5’-1a (ACG GTC GGG AAA CTA GCT CTA C)

Ds5’-2a (TCC GTT CCG TTT TCG TTT TTT AC)

Ds5’-3a (CGG TCG GTA CGG GAT TTT CC)

If no specific amplification is detected using the standard primers above, the following alternative set of Ds-complementary primers may prove to be effective:

Complementary to the 3’ end:

Ds3’-1 (CGA TTA CCG TAT TTA TCC CGT TCG)

Ds3’-2 (CCG GTA TAT CCC GTT TTC G)

Ds3’-3 (GAA AAT GAA AAC GGT AGA GGT)

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Complementary to the 5’ end:

Ds5’-1 (CCG TTT ACC GTT TTG TAT ATC CCG)

Ds5’-2 (CGT TCC GTT TTC GTT TTT TAC C)

Ds5’-3 (CGG TCG GTA CGG GAT TTT CC)

AKF Line	GABI-Kat Line