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. 2011 Dec;39(22):e148.
doi: 10.1093/nar/gkr764. Epub 2011 Sep 24.

Mobilization of giant piggyBac transposons in the mouse genome

Meng Amy Li  1 , Daniel J Turner, Zemin Ning, Kosuke Yusa, Qi Liang, Sabine Eckert, Lena Rad, Tomas W Fitzgerald, Nancy L Craig, Allan Bradley
Affiliations

Mobilization of giant piggyBac transposons in the mouse genome

Meng Amy Li et al. Nucleic Acids Res. 2011 Dec.

Abstract

The development of technologies that allow the stable delivery of large genomic DNA fragments in mammalian systems is important for genetic studies as well as for applications in gene therapy. DNA transposons have emerged as flexible and efficient molecular vehicles to mediate stable cargo transfer. However, the ability to carry DNA fragments >10 kb is limited in most DNA transposons. Here, we show that the DNA transposon piggyBac can mobilize 100-kb DNA fragments in mouse embryonic stem (ES) cells, making it the only known transposon with such a large cargo capacity. The integrity of the cargo is maintained during transposition, the copy number can be controlled and the inserted giant transposons express the genomic cargo. Furthermore, these 100-kb transposons can also be excised from the genome without leaving a footprint. The development of piggyBac as a large cargo vector will facilitate a wider range of genetic and genomic applications.

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Figures

Figure 1.
Figure 1.
PiggyBac-mediated large-cargo transposition in mouse ES cells. (a) Giant HPRT-PiggyBac constructs modified from the same BAC. (b) A scheme of competing genomic integration pathways. PBase can mediate precise excision of the giant PB from the BAC and insert it into the ES cell genome, generating cells that are resistant both to HAT and FIAU. If physical breakage of the BAC occurs and the PB ITRs are separated and random integration of the BAC can occur together with the PuroΔtk cassette. If the HPRT gene on the BAC is intact, the cells will be HAT resistant but sensitive to FIAU. (c) Transposition efficiency of different sized PB transposons and versions of the PBase. The number of transposition events was determined using massively parallel sequencing from pooled HAT and FIAU resistant clones. Asterisks indicate percentage of transposition events as a fraction of HAT and FIAU double-resistant colonies. The transposition events are assumed to be one per cell.
Figure 2.
Figure 2.
Large cargos delivered by giant PB transposons are intact. (a) Precise integration of giant PB transposons at the expected TTAA site. The chromosomal coordinates of the first T corresponding to the PB recognition site TTAA are shown (NCBI m37). The shaded sequences represent the ends of the PB ITRs. (b) Southern blot illustrating the copy number of the PB mediated largo-cargo integrations using the PB5′ITR as the detection probe. (c) Regional CGH analysis showing the gain of an extra copy of the human HPRT gene delivered by PB transposition. The red line was calculated as the running median of the Log2 value of each CGH probe to aid the visualization.
Figure 3.
Figure 3.
Stable integration of a PB transposon harboring the human FAH locus. (a) Schematic representation of the PB-FAH-62.5 construct and the selection strategy used for enriching genuine transposition events. (b) Transposition efficiency of PB-FAH-62.5 using different transfection methods and selection schemes. ‘Colonies analyzed’: the number of colonies analyzed by splinkerrette PCR for the determination of the PB ITR to genomic junction sequence. Asterisk indicates percentage of transposition events as a fraction of colonies analyzed.
Figure 4.
Figure 4.
PBase-mediated excision of giant PB transposons from the ES cell genome. (a) Genomic excision efficiency of five ES cell clones containing giant PB transposons with different cargo sizes following transfection with either HyPBase or an eGFP (control). (b) Molecular analysis of individual 6-TG-resistant colonies to evaluate fidelity of excision events. Excision of PB eliminates the PB-host junction fragment amplified in the parental lines. (c) Analysis of PB excision identified one clone with a micro-deletion (clone Dc7) following the excision of a 100-kb PB transposon. ‘n’ represents the number of 6-TG-resistant colonies with the shown sequence traces of the excision site.
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References

    1. Wade-Martins R, White RE, Kimura H, Cook PR, James MR. Stable correction of a genetic deficiency in human cells by an episome carrying a 115 kb genomic transgene. Nat. Biotech. 2000;18:1311–1314. - PubMed
    1. Hibbitt O, Wade-Martins R. Delivery of large genomic DNA inserts >100 kb using HSV-1 amplicons. Curr. Gene Ther. 2006;6:325–336. - PubMed
    1. Giraldo P, Montoliu L. Size matters: use of YACs, BACs and PACs in transgenic animals. Transgenic Res. 2001;10:83–103. - PubMed
    1. Valenzuela DM, Murphy AJ, Frendewey D, Gale NW, Economides AN, Auerbach W, Poueymirou WT, Adams NC, Rojas J, Yasenchak J, et al. High-throughput engineering of the mouse genome coupled with high-resolution expression analysis. Nat. Biotech. 2003;21:652–659. - PubMed
    1. Wallace HAC, Marques-Kranc F, Richardson M, Luna-Crespo F, Sharpe JA, Hughes J, Wood WG, Higgs DR, Smith AJH. Manipulating the mouse genome to engineer precise functional syntenic replacements with human sequence. Cell. 2007;128:197–209. - PubMed

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