The large bat Helitron DNA transposase forms a compact monomeric assembly that buries and protects its covalently bound 5'-transposon end.

Helitrons are widespread eukaryotic DNA transposons that have significantly contributed to genome variability and evolution, in part because of their distinctive, replicative rolling-circle mechanism, which often mobilizes adjacent genes. Although most eukaryotic transposases form oligomers and use RNase H-like domains to break and rejoin double-stranded DNA (dsDNA), Helitron transposases contain a single-stranded DNA (ssDNA)-specific HUH endonuclease domain. Here, we report the cryo-electron microscopy structure of a Helitron transposase bound to the 5'-transposon end, providing insight into its multidomain architecture and function.

Helraiser intermediates provide insight into the mechanism of eukaryotic replicative transposition.

Helitrons are eukaryotic DNA transposons that have profoundly affected genome variability via capture and mobilization of host genomic sequences. Defining their mode of action is therefore important for understanding how genome landscapes evolve. Sequence similarities with certain prokaryotic mobile elements suggest a "rolling circle" mode of transposition, involving only a single transposon strand.

A Helitron transposon reconstructed from bats reveals a novel mechanism of genome shuffling in eukaryotes.

Helitron transposons capture and mobilize gene fragments in eukaryotes, but experimental evidence for their transposition is lacking in the absence of an isolated active element. Here we reconstruct Helraiser, an ancient element from the bat genome, and use this transposon as an experimental tool to unravel the mechanism of Helitron transposition. A hairpin close to the 3'-end of the transposon functions as a transposition terminator.

Reprogramming triggers endogenous L1 and Alu retrotransposition in human induced pluripotent stem cells.

Human induced pluripotent stem cells (hiPSCs) are capable of unlimited proliferation and can differentiate in vitro to generate derivatives of the three primary germ layers. Genetic and epigenetic abnormalities have been reported by Wissing and colleagues to occur during hiPSC derivation, including mobilization of engineered LINE-1 (L1) retrotransposons. However, incidence and functional impact of endogenous retrotransposition in hiPSCs are yet to be established.

Brief report: impaired cell reprogramming in nonhomologous end joining deficient cells.

Although there is an increasing interest in defining the role of DNA damage response mechanisms in cell reprogramming, the relevance of proteins participating in nonhomologous end joining (NHEJ), a major mechanism of DNA double-strand breaks repair, in this process remains to be investigated. Herein, we present data related to the reprogramming of primary mouse embryonic fibroblasts (MEF) from severe combined immunodeficient (Scid) mice defective in DNA-PKcs, a key protein for NHEJ. Reduced numbers of induced pluripotent stem cell (iPSC) colonies were generated from Scid cells using reprogramming lentiviral vectors (LV), being the reprogramming efficiency fourfold to sevenfold lower than that observed in wt cells.

Sleeping Beauty transposon-based system for cellular reprogramming and targeted gene insertion in induced pluripotent stem cells.

The discovery of direct cell reprogramming and induced pluripotent stem (iPS) cell technology opened up new avenues for the application of non-viral, transposon-based gene delivery systems. The Sleeping Beauty (SB) transposon is highly advanced for versatile genetic manipulations in mammalian cells. We established iPS cell reprogramming of mouse embryonic fibroblasts and human foreskin fibroblasts by transposition of OSKM (Oct4, Sox2, Klf4 and c-Myc) and OSKML (OSKM + Lin28) expression cassettes mobilized by the SB100X hyperactive transposase.

Derivation and characterization of sleeping beauty transposon-mediated porcine induced pluripotent stem cells.

The domestic pig is an important large animal model for preclinical testing of novel cell therapies. Recently, we produced pluripotency reporter pigs in which the Oct4 promoter drives expression of the enhanced green fluorescent protein (EGFP). Here, we reprogrammed Oct4-EGFP fibroblasts employing the nonviral Sleeping Beauty transposon system to deliver the reprogramming factors Oct4, Sox2, Klf4, and cMyc.

Insertional engineering of chromosomes with Sleeping Beauty transposition: an overview.

Novel genetic tools and mutagenesis strategies based on the Sleeping Beauty (SB) transposable element are currently under development with a vision to link primary DNA sequence information to gene functions in vertebrate models. By virtue of its inherent capacity to insert into DNA, the SB transposon can be developed into powerful tools for chromosomal manipulations. Mutagenesis screens based on SB have numerous advantages including high throughput and easy identification of mutated alleles.

Generating knockout rats by transposon mutagenesis in spermatogonial stem cells.

Disrupting genes in the rat on a genome-wide scale will allow the investigation of many biological processes linked to human health. Here we used transposon-mediated mutagenesis to knock out genes in rat spermatogonial stem cells. Given the capacity of the testis to support spermatogenesis from thousands of transplanted, genetically manipulated spermatogonia, this approach paves a way for high-throughput functional genomic studies in the laboratory rat.

Comparative analysis of transposable element vector systems in human cells.

Transposon-based gene vectors have become indispensable tools in vertebrate genetics for applications ranging from insertional mutagenesis and transgenesis in model species to gene therapy in humans. The transposon toolkit is expanding, but a careful, side-by-side characterization of the diverse transposon systems has been lacking. Here we compared the Sleeping Beauty (SB), piggyBac (PB), and Tol2 transposons with respect to overall activity, overproduction inhibition (OPI), target site selection, transgene copy number as well as long-term expression in human cells.