Genetic screens using the piggyBac transposon

Abstract

Transposons are an attractive system to use in genetic screens as they are molecularly tractable and the disrupted loci that give rise to the desired phenotype are easily mapped. We consider herein the characteristics of the piggyBac transposon system in complementing existing mammalian screen strategies, including the Sleeping Beauty transposon system. We also describe the design of the piggyBac resources that we have developed for both forward and reverse genetic screens, and the protocols we use in these experiments.

Fig. 1

Catalogue of transposon designs useful for different genetic screens. They all have PB inverse terminal repeats to facilitate delivery and nested SB within it for flexibility in subsequent mobilisation (TR, terminal repeat; SA, splice acceptor; pA, polyadenylation sequence; SD, splice donor; IRES, internal ribosomal entry site).

Fig. 2

Typical genetic cross scheme for obtaining F1 experimental cohort mice carrying both the PB transposons and the transposase. The PLPB Rosa-PBase line used herein results in whole body somatic mobilisation of the transposons.

Fig. 3

Survival curves of multiple ATP2 lines. Mice with ATP2 transposons and constitutive PB transposase expression show significantly decreased survival.