The Piggybac System

The human genome consists of about 28,000 protein coding genes, numerous small RNA genes and large pieces of DNA with regulatory or unknown functions. Understanding the function of all these elements is critical to learn the human biology and diseases. However, traditional genetic methods such as gene knockout or ENU mutagenesis, are either inefficient or prohibitively expensive to be used for large-scale functional annotation of the mammalian genome.Transposable elements have been widely used as tools for transgenesis and insertional mutagenesis in lower organisms. In contrast, they haven't been commonly applied in mice and other mammals due to the lack of an efficient transposition system.We have modified piggyBac (PB), a DNA transposon from the cabbage looper moth Trichoplusia ni, and showed that it can efficiently transpose in cultured human and mouse cells as well as in mice. We show a number of technical advantages of PB acting as a mutagenesis tool. Efficient PB transposition in germline allows rapid generation of mutant animals by simple crossing. This not only bypasses expensive and technically demanding manipulations such as ES cell culture and animal surgery, which are required for classical gene knockout method, but also makes it possible to mutate genes in mammalian species with no established ES cell lines. PB insertions can be easily mapped by inverse PCR, which makes it easier to track mutations than chemical mutagenesis. PB transposon favors to hit genes genome-widely and effectively disrupts their function. Meanwhile, precise excision of PB allows reliable confimation of gene-function relationship by revertant. Application of visible markers in the PB system allows us to distinguish animals with different genotypes without PCR, which further increases mutagenesis efficiency. PB can also be used for gene trap studies to reveal expression patterns of endogenous genes. As a result, PB-mediated insertional mutagenesis permits for the first time the efficient production of mouse mutants for systematical functional analysis of the mouse genome.At the same time, we developed PB-mediated transgenic system in cultured mammalian cells and in mice. PB can carry large pieces of DNA and efficiently integrate into the host genome. Large capacity also allows PB vector to carry a visible marker along with the transgene, which makes it easier to identify the transgenic animal. The transgenes carried by PB express stably among generations. PB transposons integrate as single copies, which not only mimic endogenous genes, but also allow easy mapping of the integration sites. All together, PB system serves as an attractive transgenic tool both for cultured mammalian cells and for living animals. It can also be considered as a non-viral vector for gene therapy.Our data provide a first and critical step towards a highly efficient transposon system for a variety of genetic manipulations in mammals. We hope applications of the PB system will accelerate functional annotation of the mammalian genome, and will benefit the study of human biology and diseases.