Yergeau DA et al. (2011), Remobilization of Sleeping Beauty transposons i...

XB-ART-44507

Mob DNA 2011 Nov 24;2:15. doi: 10.1186/1759-8753-2-15.

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Remobilization of Sleeping Beauty transposons in the germline of Xenopus tropicalis.

Yergeau DA , Kelley CM , Kuliyev E , Zhu H , Johnson Hamlet MR , Sater AK , Wells DE , Mead PE .

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The Sleeping Beauty (SB) transposon system has been used for germline transgenesis of the diploid frog, Xenopus tropicalis. Injecting one-cell embryos with plasmid DNA harboring an SB transposon substrate together with mRNA encoding the SB transposase enzyme resulted in non-canonical integration of small-order concatemers of the transposon. Here, we demonstrate that SB transposons stably integrated into the frog genome are effective substrates for remobilization. Transgenic frogs that express the SB10 transposase were bred with SB transposon-harboring animals to yield double-transgenic 'hopper' frogs. Remobilization events were observed in the progeny of the hopper frogs and were verified by Southern blot analysis and cloning of the novel integrations sites. Unlike the co-injection method used to generate founder lines, transgenic remobilization resulted in canonical transposition of the SB transposons. The remobilized SB transposons frequently integrated near the site of the donor locus; approximately 80% re-integrated with 3 Mb of the donor locus, a phenomenon known as 'local hopping'. In this study, we demonstrate that SB transposons integrated into the X. tropicalis genome are effective substrates for excision and re-integration, and that the remobilized transposons are transmitted through the germline. This is an important step in the development of large-scale transposon-mediated gene- and enhancer-trap strategies in this highly tractable developmental model system.

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Species referenced: Xenopus tropicalis
Genes referenced: actl6a cntn1 myh3

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	Figure 1. Generation of a transgenic Xenopus tropicalis that expresses SB10 transposase. (a) Schematic of the pCAGGS-SB10;γcRFP construct used to develop SB (SB10) transposase-expressing transgenic frogs. The two transgenes were cloned in a tail-to-tail orientation. Not to scale. (b) Red lens in the right eye of an adult F1 transgenic frog from outcross of founder CAGGS-SB10;γcRFP 2 M. The border of the eye is indicated by the dashed white line. (c) Southern blot analysis of genomic DNA harvested from RFP-positive and control animals indicated integration of multiple copies of the CAGGS-SB10;γcRFP linear transgene. The DNA was digested with BamHI and the blot was probed with a radiolabelled SB10 cDNA probe (see schematic (a)). (d) RT-PCR analysis of SB10 expression in tadpoles. SB RNA was detected in RFP-positive tadpoles (+RFP) but not in RFP-negative (-RFP) progeny from CAGGS-SB10;γcRFP 2M. RNA from a wild-type tadpole was used as a negative control (St. 15). A mock reverse transcription reaction, without added RT, with RNA harvested from an RFP-positive tadpole (+RFP(-RT)) was used as a negative control. Primers for X. tropicalis α-actin were used as a control for RNA recovery. (e) Western blot analysis of SB transposase expression in tissues harvested from adult transgenic frogs. A monoclonal antibody to SB was used to demonstrate abundant transposase expression in the testis and liver of RFP-positive adults, but not in the RFP-negative siblings. Protein lysates prepared from tadpoles injected with SB10 mRNA at the one-cell stage were prepared at stage 15 (control lane). The blots were stripped and re-probed with a monoclonal antibody that recognizes Xenopus α-actin. PCR: polymerase chain reaction; RFP: red fluorescent protein; RT: reverse transcriptase; SB: Sleeping Beauty.
	Figure 2. Breeding strategy to generate double-transgenic hopper frogs. The F2 hopper frogs were outcrossed with wild-type animals and the progeny was scored for GFP and RFP expression. The GFP-positive/RFP-negative F3 progeny were either raised to adulthood for outcross or genomic DNA was harvested after stage 45 for molecular analyses. GFP: green fluorescent protein; RFP: red fluorescent protein.
	Figure 3. Somatic remobilization of pT2βGFP in double transgenic tadpoles. Outcross of double transgenic hopper frogs resulted in progeny that inherited both transgenes. In rare instances, we identified double transgenic tadpoles that express intense levels of the GFP transgene reporter in individual cells or in small groups of cells. The change in GFP expression seen in these somatic cells is likely to be due to sporadic remobilization of the pT2βGFP transposon and the change in GFP intensity is likely due to the influence of the local chromatin environment at the novel integration site. The region of each tadpole shown (dashed box) is indicated on the cartoon inset. (a) Tail of a double transgenic tadpole with a single muscle cell expression intense GFP (arrow). (b) Double transgenic tadpole with high-level GFP expression in a subset of cells in the brachial cartilage (arrow). The immobilized tadpole was also photographed using a dsRED filter and the two images were overlaid to demonstrate that this animal had inherited the CAGGS-SB10;γcRFP transgene (RFP expression in the lens is indicated by the white arrowhead). GFP: green fluorescent protein; RFP: red fluorescent protein.
	Figure 4. Excision and re-integration of SB transposons in the progeny of double-transgenic hopper frogs. (a) GFP expression in sibling tadpoles derived from the outcross of an 8F hopper frog. Tadpoles 1 and 2 are significantly brighter than their GFP-positive siblings (tadpoles 3, 4 and 5). Tadpole 6 is a GFP-negative tadpole. Dorsal view, with anterior facing towards the right. (b) Representative data for the outcross population of 8F hopper frogs. Table includes data from breeding four F2 (8F♂54, 8F♂55, 8F♂56 and 8F♀61) and seven F3 (8F35♂A, B, C etc.) double-transgenic hoppers with wild-type frogs. The outcross progeny were scored for GFP expression and the GFP-bright progeny were either harvested for integration site analysis or raised to adulthood and outcrossed. A range of apparent remobilization activity from 0% to 0.7% was observed in individual 8F hopper frogs, with an average rate of two remobilization events per thousand GFP-positive progeny (0.2%). (c) Southern blot analysis of genomic DNA harvested from the progeny of double transgenic 8F hopper frogs. Genomic DNA was digested with BglII and the blot was probed with a radiolabelled GFP cDNA probe. DNA harvested from tadpoles in lanes 3, 4 and 5 have the same banding pattern as the parental pT2βGFP 8F founder line. Lanes 1 and 2 show example of remobilization of an SB transposon. The dashed arrow indicates the change in the mobility of the transposon-harboring BglII fragment. Lane 6 contains DNA from GFP-negative siblings. GFP: green fluorescent protein; SB: Sleeping Beauty.
	Figure 5. Integration site analysis of remobilized SB transposons. (a) Schematic representation of the 8F donor locus showing the predicted orientation of the trimeric concatemer in scaffold 57. This injection-mediated integration event occurred by a non-canonical mechanism. (Not to scale.) (b) Schematic representation of the novel integration event in the remobilized tadpole shown in Figure 4b. EPTS LM-PCR was used to clone the sequence flanking the 5' end of the pT2βGFP transposon and the 5' and 3' flanking sequences were verified using PCR primers designed to the scaffold sequence. The novel integration event occurred on the same scaffold (57 at position 2544323 bp) of the Joint Genome Institute X. tropicalis genome sequence assembly v4.1 as the 8F transposon donor locus (57:2456981) and represented a local hop. The sequence of the SB transposon and genomic DNA junctions (arrows) indicated that the remobilization event occurred via a canonical transposition reaction. The pT2βGFP transposon is flanked by the expected TA dinucleotide target site duplication (TSD; bold underlined), and the transposon is inserted precisely, without any flanking plasmid sequence from the donor site. The genomic DNA sequence of scaffold 57 is capitalized and the transposon sequence is in lowercase italics. (Not to scale.) (c) The preferred sequence for SB transposon re-integration in the X. tropicalis genome. Weblogo analysis http://weblogo.berkeley.edu for the five base pair sequence flanking the TA target site. The relative size of the letters indicates the strength of the information on the y-axis, with the maximum indicated by two bits. The table shows the base distribution of the pT2βGFP transposon re-integration target sites. EPTS LM-PCR: extension primer tag selection linker-mediated polymerase chain reaction; PCR: polymerase chain reaction; SB: Sleeping Beauty.
	Figure 6. Schematic representation of remobilized SB transposons in the X. tropicalis genome. (a) Local hopping on X. tropicalis chromosome 6 depicts the integration sites for eight local (< 200 kb) remobilization events (hops). (Not to scale.) This region of X. tropicalis chromosome 6 is syntenic with human chromosome 7. The Vista http://genome.lbl.gov/vista alignment shows regions of homology between the frog and human genomic sequences; pink represents non-coding regions and blue represents conserved exons. The position of the 8F donor concatemer is indicated by the grey box. The remobilized transposition events are depicted by the grey triangles. The position and orientation of predicted genes near the 8F locus are depicted in the lower section of the panel. (b) Schematic representation of the X. tropicalis chromosomes indicating the distribution of remobilized SB transposons. The parental 8F donor site is on chromosome 6 (thick line). The predicted loci of the remobilized transposons are depicted by the thin black lines. Approximately 80% of the remobilization events occur near the donor locus (local hopping), and the remaining 20% are distributed randomly throughout the genome. SB: Sleeping Beauty.
	Figure 7. Transposon hopping from the 7M donor locus. (a) The outcross progeny from nine 7M hopper adults were scored for changes in GFP intensity indicative of transposon remobilization. The 7M donor locus contains a concatemer of approximately eight to ten pT2βGFP transposons on chromosome 10. The remobilization frequency is expressed as a percentage of GFP bright tadpoles observed in the GFP-positive outcross population. 7M hopper frogs 7M♂9 and 7M♂11 are 'functionally homozygous' for both the transposon substrate allele and the transposase enzyme transgene (see text for details), and display higher remobilization activity (approximately 1.8%) than their 'heterozygous' male hopper littermates (7M♂1, 7M♂2, 7M♂3, 7M♂5, 7M♂14, 7M♂20; approximately 0.44%). Outcross of 7M hopper 7M♀25 produced a remobilization rate of > 4%. (b) Schematic representation of the X. tropicalis chromosomes indicating the distribution of remobilized SB transposons. The parental 7M donor site (thick line) is located on scaffold 38 which maps to chromosome 10. Remobilization of discrete transposons away from the 7M donor locus is represented by the grey arrows. (Not to scale.) (c) Fluorescence in situ hybridization of metaphase chromosomes verifies that the 7M parental donor locus is located near a telomere of X. tropicalis chromosome 10. GFP: green fluorescence protein; SB: Sleeping Beauty.

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