Modelling leukemia in the mouse: novel strategies in genome engineering

Testa, Guiseppe (2002). Modelling leukemia in the mouse: novel strategies in genome engineering. PhD thesis The Open University.



The work of this thesis presents novel genome engineering strategies for assembling complex targeting vectors for functional studies in the mouse. With the objective of establishing a conditional mouse model of the human acute leukemia associated with the t(4;ll)(q21;q23) translocation, an approach based on the Cre-loxP technology was chosen, and two mouse lines and one ES cell line were engineered starting from BACs.

For the Af4 mouse line, a combinatorial mutant allele was designed, which supports Cre-driven interchromosomal translocation, can result in a knock-out or hypomorphic allele (also amenable to conditional gene repair strategies) and can report endogenous expression of the gene through |3-galactosidase staining. The work utilised several variations of the ET cloning methodology and served to establish their feasibility for BAG engineering as a novel approach to the generation of complex mouse knock-in/knock-out targeting vectors. Particularly noteworthy was the application of ET technology to the direct subcloning of very large BAG fragments.

The Mll targeting construct constitutes the first example of a BAG based, very large knock-in vector (about 70 kb) which was successfully used to target the endogenous Mll locus by homologous recombination in mouse ES cells. The construct was engineered via ET recombination approaches. The major novelty consists in the targeting design which can simultaneously mutate two sites of the gene located very far from each other. Combined with the power of site specific recombinases (SSRs) technology, this opens the way to establishing complex, versatile mouse lines with only one round of ES cell targeting.

For the second mouse line, a BAG transgenesis approach was chosen to place Cre recombinase under the control of the Ikaros gene, in a new configuration which can yield either constitutive or regulated Cre activity. Several BAG modifications were applied, and the usefulness of Tnpl recombinase in BAG engineering was established.

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