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Pioneering New Genetic Tools & Approaches

piggyBac: A Powerful Genetic Tool

Forward genetics have played a pivotal role in our understanding of modern biology in lower organisms. However, the lack of comparable genetic tools in mammals has impaired our ability to gain understanding of many aspects of mammalian biology and disease. The current approaches for gene inactivation in the mouse, the mammalian model of choice, have been limited to targeted gene knockouts or chemical mutagenesis, which is either prohibitively expensive to scale up or largely intractable due to technical inefficiency. Furthermore, there is really no efficient approach for transgenesis in most vertebrates and mammals.

One of the most useful approaches for genetic manipulation is the use of transposons for generating transgenic organisms and for producing insertional mutants. Ever since the discovery of transposons in maize by Barbara McClintock in the late 40s (McClintock, 1950), generations of geneticists have attempted to develop an efficient transposon system for mammals. Transposons including members from the Tc1/Mariner family such as Sleeping Beauty (SB), have previously been used in the mouse (Ivics et al., 1997; Luo et al., 1998).

Although they have been used for insertional mutagenesis in the mouse germline and somatic cells, their general application for mouse genetics has been limited for several reasons: 1) subsequent to remobilization, new insertions heavily concentrate near the original site, 2) transposition events occur at low efficiencies, and 3) limited capacity for carrying large inserts, and 4)introducing chromosomal deletions during transposition.

In searching for an efficient tool for genetic manipulations in mammals, we have modified thepiggyBac (PB) transposable element from the cabbage looper moth Trichoplusia ni to generate a binary PB transposon system for mammalian cells and organisms (Ding et al., 2005). It requires a non-autonomous PB transposon cassette for delivering the exogenous gene(s) of interest, and a transgene expressing the PBtransposase enzyme (PBase) for inducing transposition in mammalian cells.

Using this system, we have shown that thePB carrying multiple transgenes can efficiently transpose in the mouse germline. In contrast to the unstable expression exhibited by traditional concatamer transgenes or viral transgenes, the PBtransgenes exhibit stable inherence and expression. Thus,PB provides highly efficient means to generate single copy transgenes, which can be genetically traced with a visible marker. Thus, the mammalian PB system provides a powerful and universal tool for genetic manipulations in mammals.

Binary piggyBac transposon system for mammalian genetic manipulation. A) Diagram showing mice carrying PBtransposon (PB), PB transposase (PBase), or both (PB + PBase). B) The mice can be identified by coat color from(Agouti) expression in the PBase transgenic mouse or fluorescence due to RFP expression in the PB construct. C) New PB insertions can be identified by the differential expression of the RFP marker that is dependent on the site of chromosomal insertion. D) Mouse lines carrying new PB insertions from a transposition. E) Diagram of a heterozygous mating. F) Heterozygous and homozygous progeny can be easily distinguished by the extent of their fluorescence due to single or double copy expression of the RFP gene.