High-Resolution Genome-Wide Mapping of Transposon Integration in Mammals

Abstract

The Sleeping Beauty (SB) transposon is an emerging tool for transgenesis, gene discovery, and therapeutic gene delivery in mammals. Here we studied 1,336 SB insertions in primary and cultured mammalian cells in order to better understand its target site preferences. We report that, although widely distributed, SB integration recurrently targets certain genomic regions and shows a small but significant bias toward genes and their upstream regulatory sequences. Compared to those of most integrating viruses, however, the regional preferences associated with SB-mediated integration were much less pronounced and were not significantly influenced by transcriptional activity. Insertions were also distinctly nonrandom with respect to intergenic sequences, including a strong bias toward microsatellite repeats, which are predominantly enriched in noncoding DNA. Although we detected a consensus sequence consistent with a twofold dyad symmetry at the target site, the most widely used sites did not match this consensus. In conjunction with an observed SB integration preference for bent DNA, these results suggest that physical properties may be the major determining factor in SB target site selection. These findings provide basic insights into the transposition process and reveal important distinctions between transposon-and virus-based integrating vectors.Approximately half of the mammalian genome is derived from ancient transposable elements. Although the two general types of transposable elements, (DNA) transposons and retrotransposons, are often regarded as "selfish DNA parasites" or "junk DNA," their frequent movement in and out of host cell chromosomes has played a significant role in genome diversification and evolution. Members of the Tc1/mariner family of DNA transposons are extremely widespread in nature (44) and can function independently of species-specific host factors (29,56). Although the vast majority of elements present in vertebrate genomes are nonfunctional (14, 32), an active Tc1-like element called Sleeping Beauty (SB) was recently reconstructed from ancient transposon fossils found within fish genomes (20).SB elements transpose by a cut-and-paste mechanism that requires the sequence-specific binding of the SB transposase to the transposon ends (25). This transposition process involves the precise excision and reintegration of the transposon from one DNA site to another site, which invariably contains a TA dinucleotide that is duplicated upon insertion. The transfer of DNA strands at the insertion site is mediated by the transposase catalytic core domain, which contains a conserved DDE motif shared by a large group of recombinase proteins, including the V(D)J recombinase and retrovirus integrases (44). SB is capable of efficient transposition in a variety of cell types (24), including human, mouse, and fish cells, and is an emerging tool for genetic research on vertebrates, with potential applications for transgenesis (22), functional genomics (1, 5-9, 11, 12, 16, 18, 19, 21, 33), and human gene therapy...

Engineering of PEDF-Expressing Primary Pigment Epithelial Cells by the SB Transposon System Delivered by pFAR4 Plasmids

“…Furthermore, SB transposon integrations occur in genomic safe harbors, defined by five criteria32, 33 (Figure 7C), at about 25% (Figure 7D). These results are in good agreement with numerous previous studies, which established a close-to-random insertion profile for the SB system in human cells 34, 35, 36, 37…”

“…In contrast to the genomic integration profile of retroviral systems and other transposon systems ( piggyBac and Tol2 ), which show integration preferences for actively transcribed genes,51, 52, 53 SB -based integration is random 37, 54. Compared to computationally generated control datasets, SB100X -mediated genomic integration of the PEDF gene delivered by the pFAR4 vector into primary human RPE cells showed a close-to-random profile.…”

Pig transgenesis by Sleeping Beauty DNA transposition

“…One of the advantages of SB transposon vectors is the random integration profile (Liu et al 2005;Staunstrup et al 2009;Yant et al 2005). Thus, SB vectors are less likely to target genes but may be more vulnerable to transcriptional silencing when inserted in heterochromatin.…”

“…SB was genetically reconstructed from fossil elements in the genome of salmoid fish (Ivics et al 1997) and is actively mobilized in cells derived from a variety of vertebrate species . SB-derived vectors efficiently jump from plasmid DNA introduced into cells to chromosomal DNA and are inserted without particular preference for actively transcribed genes (Liu et al 2005;Yant et al 2005). The element has been explored for the use in therapeutic gene transfer (Izsvak and Ivics 2004;Liu et al 2006;Mates et al 2009;Mikkelsen et al 2003;Ohlfest et al 2004;Singh et al 2008;Yant et al 2000) and is widely used as a tool in animal transgenesis.…”

Effective Expression-Independent Gene Trapping and Mutagenesis Mediated by Sleeping Beauty Transposon

“…Accordingly, it is used for long-term expression in transgenesis and insertional mutagenesis in vertebrates Largaespada et al, 2005). In addition, an analysis of 1336 inserts in primary and cultured mammalian cells has demonstrated that SB integration exhibits less preference for transcriptional units and/or their promoters compared with retroviruses (Yant et al, 2005). Therefore, the SB system appears to be an ideal non-viral vehicle for production of transgenic animals and potential genome-wide mutagenesis due to its relative high activity, large cargo capacity and less integration site preference.…”