Cre/Lox Deletions

Knocking-out tandem gene arrays.

The example below demonstrates how pDs-Lox lines can be used to knock-out all three members of a three-gene tandem array. The same strategy could also be used to knockout any number of tandemly repeated genes.
 

Step 1.  Isolate a plant carrying a T-DNA insertion within one member of the gene family.
       

       

Step 2.  Mobilize the Ds element by crossing to a plant expressing the Ac transposase. Use Hygromycin to select for transposon excision. Use PCR to screen for Ds insertions within other members of the tandem gene array. This two step strategy can be used to make double-mutant combinations. If a triple-mutant is desired, then step number three described below is necessary.

       


Step 3.  Catalyze deletion of the chromosomal region that lies between the two LoxP sites by crossing the line above to a plant that expresses the Cre recombinase. Identify plants carrying deletions by screening for luciferase expression or by using PCR.

Are the LoxP sites functional?

We have tested the functionality of the LoxP sites present in pDs-Lox and found that they are indeed able to direct deletion of intervening DNA sequences.  This experiment involved taking one of the lines from our pDs-Lox population and crossing it with a plant that expresses the Cre recombinase under the control of the 35S promoter.  Progeny from parent plants that were hemizygous for both the pDs-Lox insertion and the Cre expression construct were analyzed by PCR to find individuals that had undergone Cre/Lox mediated deletion.   DNA sequencing was then used to confirm that the deletion events were specific to the LoxP sites.  (Krysan et al, unpublished data). 

It should be noted that this experiment did not involve a transposed Ds element.  Instead, we simply tested the ability of the LoxP sites to direct recombination in the context of the intact pDs-Lox vector prior to Ds hopping.  The importance of this experiment is that it demonstrates that the LoxP sites present in pDs-Lox are indeed functional and can direct deletion in the context of the Arabidopsis genome.  We are in the process of testing the efficiency of Cre/Lox mediated deletion of Arabidopsis genomic sequences using this system.  A recent publication from the laboratory of Dr. Nina Fedoroff describes the use of a similar vector system for performing Cre/Lox deletions.  This paper should be of interest to people exploring the use of Cre-Lox mediated deletions in Arabidopsis.  Click here to open a new window featuring that publication.

The Cre expression line that we used in these experiments is from the Columbia ecotype.  We are in the process of further characterizing these lines before we make them publicly available.  We will make an announcement on this web site when the Cre expression lines are ready for distribution.