piggyBac Transposon-Based Insertional Mutagenesis in Mouse Haploid Embryonic Stem Cells

Abstract

Forward genetic screening is a powerful non-hypothesis-driven approach to unveil the molecular mechanisms and pathways underlying phenotypes of interest. In this approach, a genome-wide mutant library is first generated and then screened for a phenotype of interest. Subsequently, genes responsible for the phenotype are identified. There have been a number of successful screens in yeasts, Caenorhabditis elegans and Drosophila. These model organisms all allow loss-of-function mutants to be generated easily on a genome-wide scale: yeasts have a haploid stage in their reproductive cycles and the latter two organisms have short generation times, allowing mutations to be systematically bred to homozygosity. However, in mammals, the diploid genome and long generation time have always hampered rapid and efficient production of homozygous mutant cells and animals. The recent discovery of several haploid mammalian cell lines promises to revolutionize recessive genetic screens in mammalian cells. In this protocol, we describe an overview of insertional mutagenesis, focusing on DNA transposons, and provide a method for an efficient generation of genome-wide mutant libraries using mouse haploid embryonic stem cells.

Transposons As Tools for Functional Genomics in Vertebrate Models

“…Importantly, haploid ESCs isolated from androgenetic embryos have been demonstrated to support the generation of germline-modified mice via intracytoplasmic injection into oocytes [77, 78]. Moreover, haploid ESCs can serve as a convenient tool to generate homozygous mutants by transposon mutagenesis on a genome-wide scale for functional gene studies [79]. …”

Endogenous Transposase Source in Human Cells Mobilizes piggyBac Transposons

“…4 The T. ni PB transposon has 13-bp ITRs at its ends, inserts into TTAA target sequences, and is transpositionally active in a wide range of cell types and species. Thus, the PB transposon has become a widely used, popular tool for a variety of applications including animal transgenesis, 5 mutagenesis screens to functionally annotate genes in vitro 6,7 and in vivo, 8,9 induced pluripotent stem cell reprogramming, 10,11 and gene therapy. [12][13][14][15][16] Transposons are not only useful tools for genetic engineering, but have also played a key role in the evolution of novel gene functions in nature.…”

Hunting Viral Receptors Using Haploid Cells

“…Other forms of mutagenesis include transposon-based insertional mutagenesis, which has been successfully employed in haploid ES cells (63; 70; 74), and chemical mutagenesis. The latter relies on a chemical mutagen such as N-ethyl-N-nitrosourea (ENU), and has the potential to efficiently, and with reduced bias, mutagenize the haploid genome (75).…”