Ds Transposon

• Using the Ds element present in pDS-Lox
• pDS-Lox does not express any transposase protein
• The Ds element in pDs-Lox is completely stable in the absence of transposase
• pDS-Lox lines must be crossed to a plant expressing Ac transposase in order to mobilize the transposon

Figure 1.  pDs-Lox vector. 

This vector was used to create the new Wisconsin Collection of T-DNA lines. Everything between the two Ds border sequences "hops" out of the T-DNA vector during transposition. The Ds element present in pDS-Lox will not transpose until the line carrying pDS-Lox is crossed to a plant that expresses the Ac transposase protein. T-DNA lines created using pDS-Lox can therefore be used directly for reverse-genetic analysis of single genes, and the insertions will remain stable.  Click here to view a detailed legend to Figure 1 in a new window.  pDs-Lox is a derivative of the plasmid pED204 (Medberry et al, 1995).  To view the annotated sequence of pDs-Lox  click here.

Figure 2.  Using pDS-Lox as a Ds Launch-Pad for local mutagenesis. 

By crossing a T-DNA line carrying this vector to a plant that expresses the Ac transposase protein, one can cause the Ds element to transpose to linked regions of the genome. The structure of the two DNA elements that result from the excision of the Ds transposon are shown. Excision of the Ds element can be positively selected for using Hygromycin resistance. PCR can then be used to identify Ds insertions that have occured in the region of interest.

Is the Transposon functional?

We originally tested the functionality of the Ds element present in pDs-Lox using plants from the T1 generation and found that it is indeed able to transpose.  This experiment involved taking lines from our pDs-Lox population and crossing them with a plant that expresses the Ac transposase under the control of the 35S promoter.  We selected hygromycin resistant progeny from parent plants that were hemizygous for both the pDs-Lox insertion and the Ac expression construct.   TAIL PCR was then used to map the locations of several transposed Ds elements (Krysan et al, unpublished data).  These results demonstrated that the Ds element present in pDs-Lox is a functional transposon.

More recently we have been testing for transposon activity using plants grown from T2 seeds, which is what one obtains from the ABRC.  Our initial survey of seven independent WiscDsLox lines has indicated that only two of them are able to support efficient Ds element transposition.  These results suggest that the Ds element present in many of the WiscDsLox lines may be subject to some type of silencing mechanism that prevents efficient transposition.  We are currently attempting to better characterize the mechanism responsible for this apparent transposon silencing.  For the time being, we are encouraging users of the WiscDsLox collection to perform a small scale pilot study with a given line in order to determine if it is able to support transposition.  If the transposon in able to efficiently mobilize, then one can continue with the process of identifying large numbers of independent transpositions.  If the element does not mobilize, then we would suggest searching for a different launch pad line in the same genomic region.

We are currently in the process of developing and testing a modified launch pad vector that should not be subject to the transposon silencing that we observe with the pDsLox vector.  Updates on the progress of this work will be provided on this website.