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1.
Fig. 4.

Fig. 4. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Transposition of TcBuster variants. The frequency of transposition of several forms of TcBuster is compared with the frequency of piggyBac. TcBuster is the ORF from Tribolium, TcBusterCO is a version codon-optimized for expression in mammalian cells, and TcBusterCO V596A contains an amino acid change based on a hyperactive mutant of the closely related AeBuster1.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
2.
Fig. 1.

Fig. 1. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

SPINON transposition in mammalian cells. The transposition of piggyBac and SPIN elements containing blasticidin resistance genes from transfected plasmids to the HeLa genome was measured in the presence and absence of transposase expressed from a CMV promoter by selection for blasticidin-resistant cells. The frequency of SPIN transposition compared with the frequency of piggyBac transposition is set as 1.0.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
3.
Fig. 2.

Fig. 2. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

TcBuster transposition in mammalian cells. The transposition of TcBuster, hyperactive Sleeping Beauty, and piggyBac elements containing antibiotic resistance genes from transfected plasmids to the HeLa genome was measured in the presence and absence of transposase by selection for antibiotic-resistant cells; the frequency of transposition of TcBuster transposition is compared with the frequency of a hyperactive Sleeping Beauty (HSB16) (19) and piggyBac.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
4.
Fig. 6.

Fig. 6. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Distribution of Sleeping Beauty, piggyBac, TcBusterCO, and SPINON insertions on the human genome with respect to transcription start sites. Integration sites near transcription start sites were compiled onto a common transcription start site, and the proportions were mapped. The x axis shows the distance from the transcription start site, and the y axis shows the percentage of integration sites in each interval.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
5.
Fig. 3.

Fig. 3. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

TcBuster transposition in mammalian cells. The transposition of TcBuster, hyperactive Sleeping Beauty, and piggyBac elements containing antibiotic resistance genes from transfected plasmids to the HeLa genome was measured by selection for antibiotic-resistant cells; the frequency of transposition of TcBuster transposition was compared with the frequency of a hyperactive Sleeping Beauty (HSB16) and piggyBac using different amounts of transposase expression plasmid and the transposon-containing donor plasmid.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
6.
Fig. 7.

Fig. 7. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

TcBusterCO transposition in yeast. (A) Integration assays that measure the transposition of a TcBuster transposon from a plasmid into the host genome by assaying plasmid-free cells for the presence of a transposon-encoded marker are shown. The transposase is expressed from a plasmid GALS promoter and thus, is induced in the presence of galactose. TcBusterCO V596A has an amino acid change comparable with the hyperactive AeBuster1 V597A mutant. (B) Excision assays that measure the excision of a TcBuster transposon from a donor site in a plasmid URA3 gene by following reversion from Ura to Ura+ are shown. The transposase is expressed from a plasmid GALS promoter and thus, is induced in the presence of galactose. TcBusterCO V596A has an amino acid change comparable with the hyperactive AeBuster1 V597A mutant.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
7.
Fig. 8.

Fig. 8. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

hAT Buster element transposition in vitro. The products of in vitro transposition reactions using end-labeled oligonucleotides and circular plasmid targets are displayed on agarose gels. (A) Substrates, steps, and products of transposition. In SEJs, a single transposon end oligonucleotide joins to the target plasmid, giving a nicked circle; in DEJs, two transposon end oligonucleotides have joined to the target plasmid, giving a linear species. (B) Target joining and coupled cleavage using TcBusterCO transposase and end oligonucleotides as indicated are shown. (C) Target joining and coupled cleavage SPINON and end oligonucleotides as indicated are shown.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
8.
Fig. P1.

Fig. P1. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Transposons for genome engineering. Transposons are DNA segments bounded by special sequences at their termini (black triangles). At these terminal sites, the transposon-encoded transposase enzyme binds the transposon, makes double-strand breaks in the DNA to cut the transposon away from the donor site, and then inserts it into a target DNA. Two plasmid systems, which can be introduced into mammalian cells by transfection, are often used for genome engineering to allow separate manipulation of the transposon and transposase. Transposons can serve as vectors to introduce genetic material for many purposes into a target genome. Here, your favorite gene (YFG) is the material being introduced. Two highly active transposons with target site selectivities, TcBuster and SPIN, are described here, which expand the toolkit available for genome engineering.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
9.
Fig. 5.

Fig. 5. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Distribution of Sleeping Beauty, piggyBac, TcBusterCO, and SPINON insertions on the human genome with respect to the indicated genomic features. (A) Integration frequency near selected genomic features. (B) Integration frequency near bound proteins and modified histones mapped using the ChIP-Seq method. In both A and B, integration site datasets for Sleeping Beauty (SB), piggyBac (PB), TcBusterCO (TCB), and SPINON (SPIN) are indicated by the columns, and genomic features or ChIP-Seq datasets are indicated by the rows (the latter were calculated over 10-kb windows). The departure from random distribution is indicated by colored tiles (key at bottom), and differences from random placement were scored using the ROC area method described in the work by Berry et al. (26). In A, blue indicates that insertions are depleted compared with random. Red indicates features where insertions are enriched compared with random. Gray indicates that the distribution is random. In B, yellow and blue are used to indicate depletion or enrichment. A detailed explanation of the variables studied can be found in the work by Ocwieja et al. (58) or at http://microb230.med.upenn.edu/assets/doc/HeatMapGuide_v12_formatted.doc.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.
10.
Fig. 9.

Fig. 9. From: A resurrected mammalian hAT transposable element and a closely related insect element are highly active in human cell culture.

Mechanism of Buster element transposition. (A) TcBuster transposition occurs through a hairpin intermediate. The products of transposition reactions over time use TcBusterCO transposase, a 3′ end-labeled oligo in which the transposon end is flanked by donor DNA and divalent metal are displayed (A) on a denaturing acrylamide gel, (B) a native agarose gel, and (C) a denaturing agarose gel. SEJ and DEJ products as described in Fig. 8 are observed on the native gel. On the denaturing gel, a 64-bp fragment containing the top strand of the transposon (63 nt) and 1 nt from the flanking donor DNA (reflecting that the initiating nick occurs 1 nt from the 5′ transposon end into the flanking donor DNA) and flanking donor 219-nt DNA hairpin species containing 110 nt of the bottom strand and 109 nt of top strand increase over time. (B) Coupled cleavage and strand transfer. The products of the reactions in A are displayed on a native agarose gel. (C) The 3′ end of the transposon is attached covalently to the target DNA. The products of reactions using either TcBusterCO or SPINON transposases as indicated and their cognate transposon ends labeled on the 5′ end of the bottom strand are displayed on a denaturing agarose gel. The covalent linkage of the labeled transposon end and target DNA in the reaction product shows that the 3′OH transposon end joined to the target DNA.

Xianghong Li, et al. Proc Natl Acad Sci U S A. 2013 February 5;110(6):E478-E487.

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