Generation and analysis of transgenic X. tropicalis expressing SB10 transposase
A transgenic X. tropicalis
line was engineered to express the SB
10 transposase under the control of a synthetic regulatory element, chicken β-actin promoter coupled with a cytomegalovirus enhancer (CAGGS [26
]. To track the inheritance of the SB
10 transgene, a X. laevis
γ1 crystallin-red fluorescent protein (RFP) [28
] reporter was cloned downstream of the CAGGS-SB
10 transgene in a head-to-head orientation (Figure ). The presence of the linked γ1 crystallin-RFP reporter allows screening for the CAGGS-SB
10 transgene based on the presence of red eyes (Figure ). We used the simple linear plasmid DNA injection method described by Etkin and Pearman to generate the transgenic SB
transposase-expressing frogs [29
]. Injected embryos were scored for the presence of RFP expression in the lens, and RFP-positive tadpoles (27 RFP-positive from 570 injected, 4.7%) were raised to adulthood. A single founder (CAGGS-SB
10;γcRFP 2 M), from a total of five animals outcrossed to date, was identified. Outcross of male founder CAGGS-SB
10;γcRFP 2M with a wild-type female resulted in 779 RFP-positive tadpoles from a total of 3,333 offspring (23.4%). The non-Mendelian inheritance of the transgene indicates that the germline of the CAGGS-SB
10;γcRFP 2M founder was mosaic for the transgene. Subsequent outcross of F1
animals derived from CAGGS-SB
10;γcRFP 2M resulted in the expected 50% of the progeny expressing the dominant lens-specific RFP reporter (in a representative F1
outcross there were 239 RFP-positive tadpoles from a total of 479, 49.9%). Southern blot analysis of RFP-positive tadpoles indicated that several copies of the transgene were integrated at a single locus in the founder (Figure ). Reverse transcriptase (RT)-PCR and Western blot analyses were used to verify that SB
10 transposase was expressed in the transgenic line. RT-PCR analysis showed that RFP-positive tadpoles at stage 40 [30
] express mRNA encoding the SB
transposase enzyme (Figure ). As expected, sibling tadpoles that did not express the RFP reporter in the lens were also negative for SB
10 mRNA expression. In adults, robust expression of SB
transposase was detected in protein lysates prepared from testes harvested from RFP-positive male frogs, but not from RFP-negative animals (Figure ). SB
10 is also expressed in the liver of the transgenic frogs, but not in the RFP-negative littermates.
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 (more ...)
βGFP 8F founder harbors two independently-segregating alleles: a concatemer of three SB
transposons integrated at a single locus on scaffold 57, at base number 2456981 (57
:2456981) of the JGI X. tropicalis
genomic sequence v4.1 assembly, and another allele with a single-copy transposon integration [6
]. Thus, the F2
hopper frogs inherited either one, or both, of the 8F integration events. Southern blot analysis of progeny from 8Fhopper♂35 indicated that this double-transgenic hopper had inherited the trimeric concatemer of pT2
βGFP on scaffold 57 alone. Double-transgenic (RFP+/GFP+) progeny (F3
) from the outcross of 8Fhopper♂35 were raised and outcrossed, and the resulting progeny (F4
) were analyzed for modification of the parental pT2
βGFP locus (Figure ).
Observation of the GFP expression in the hopper outcross populations indicated that, in most cases, the GFP expression of the progeny was identical to that of the 8F founder, suggesting that the parental SB transposon locus was intact. In a small number of the outcross progeny, we observed markedly different GFP expression in either small populations of cells within the tadpole (Figure ) or in whole tadpoles (Figure ; 40 from 20,015 GFP-positive tadpoles). We reasoned that the change in GFP expression might result from the modification of the parental pT2βGFP locus in the remobilized progeny. Embryos with small subsets of cells with increased GFP intensity likely represent stochastic transposase activity in somatic tissues (somatic remobilization (Figure )). An organism-wide change in GFP intensity (Figure ) likely represents modification of the parental transposon donor locus during gametogenesis that is passed on to the resulting progeny. Remobilization of a transposon from the donor locus to a novel site will likely alter the local epigenetic environment of the transgene, and also subject the re-integrated transposon to the influence of nearby gene regulatory sequences that differ from the parental locus.
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 (more ...)
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 (more ...)
Genomic DNA harvested from GFP-positive progeny from double transgenic (pT2βGFP8F:CAGGS-SB10;γcRFP) 8F hopper frogs was analyzed by Southern blot. Digestion of genomic DNA from pT2βGFP 8F tadpoles with BglII resulted in three bands when the blot was hybridized with a GFP probe (Figure ). Changes in the Southern blot hybridization pattern were used to determine whether the parental concatemer had been altered by expression of the SB transposase. Analysis of progeny from the outcross of F3 double transgenic hopper frogs with wild-type animals indicated that most of the progeny had inherited the unaltered pT2βGFP 8F parental concatemer (Figure ; lanes 3, 4 and 5). Examples of germline remobilization of the pT2βGFP transposon from 'GFP-bright' tadpoles (Figure ; tadpoles 1 and 2) harvested from the outcross of 8Fhopper♂58 are shown in Figure (lanes 1 and 2, dashed arrows). This data indicates that, as predicted, the GFP-bright individuals in the outcross population of the hopper frogs represent tadpoles that have modified the parental transposon donor locus. Thus, remobilized animals can be identified in the outcross population by simply observing the tadpoles for changes in GFP intensity. Outcross of eleven 8F hopper double transgenic frogs indicated that the frequency of remobilized progeny varied from 0.07% to 0.71% (Figure ). The variation in the remobilization activity between individual hopper frogs likely reflects subtle differences in epigenetic modification of the substrate and enzyme transgenes in each animal that may alter the activity of the excision and reintegration reactions.
Analysis of the cloned flanking sequences of the parental locus (57
:2456981) from the remobilized tadpoles (Figure ; lane 1 and 2) showed no sequence change, indicating that the remobilized transposon was excised from within the donor concatemer (data not shown). Extension primer tag selection linker mediated-PCR (EPTS LM-PCR) and standard genomic PCR [6
] were used to clone the integration sites of the novel bands. The re-integration event from tadpole 8Fhopper♂58-1 had occurred on the same scaffold as the parental integration site, and thus represented a 'local hop' (Table and Figure ; tadpole 8Fhopper♂58-1). Genomic PCR and sequencing was used to verify both the 5'- and 3'-ends of the novel insertion site. The integration site of the remobilized pT2
βGFP transposon is at 57
:2491386 and is 34,405 bp away from the parental locus on chromosome 6 (Figure ). Sequence analysis of the integration site indicated that the remobilization event was catalyzed by a canonical transposition event. That is, the transposon inserted precisely at the predicted boundary of the indirect repeat/direct repeats (IR/DRs). Furthermore, the integrated transposon is flanked by the expected TA dinucleotide target site duplication catalyzed by SB
]. Thus, unlike the co-injection method used to generate the pT2
βGFP founder lines that results in unexpected concatemer formation (Figure , [6
]), the remobilization events catalyzed by re-expression of SB
transposase are via canonical transposition (Figure ). To date, we have identified 40 remobilization events, based on differences in GFP expression intensity, from 20,015 GFP-positive tadpoles from the outcross of 8F hopper frogs (Figure ). Southern analysis has confirmed excision and re-integration of a SB
transposon from the parental locus and yields an apparent remobilization frequency of approximately 0.2%.
Integration site analysis for remobilized progeny from 8F hoppers.
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 (more ...)
Pre-sorting tadpoles based on GFP intensity may underestimate the total remobilization activity if the re-integration event resulted in GFP expression that was not markedly different from the parental expression. To test this, we outcrossed a double transgenic hopper frog (8Fhopper♂51) and analyzed all of the GFP-positive progeny by Southern blot. The 8Fhopper♂51 frog inherited both of the pT2βGFP transposon alleles from the 8F founder. The progeny from this 8Fhopper♂51 outcross displayed GFP expression patterns and intensities that were indistinguishable from that of the parental alleles (data not shown). From the 677 GFP-positive progeny analyzed by Southern blot, we identified four excision-only events and two remobilizations. Samples of genomic DNA where changes were evident by Southern blot analysis were used in EPTS LM-PCR to clone the integration site of the remobilization events. In this experiment, the remobilization frequency was 0.3% (two remobilization events out of 677 GFP-positive tadpoles). These data indicated that the actual remobilization frequency may be somewhat higher than that estimated by simple visual inspection of the GFP-positive progeny. The observed rate of excision-only events in this outcross population was 4 out of 677, that is, 0.6%.
Scoring the outcross progeny of hopper frogs for changes in GFP intensity may also overestimate the remobilization frequency, as this method may not distinguish between remobilization events and excision-only events. We analyzed 25 GFP-bright tadpoles from the outcross of 8F and 7M (see below) hopper frogs, by Southern blot analysis and by cloning the novel insertion sites by EPTS LM-PCR. Only one GFP-bright tadpole had an excision-only modification of the parental transposon donor locus (4%); 24 GFP-bright tadpoles (96%) had re-integration events that were evident by novel bands on the Southern blot and by cloning the sequences flanking the canonical re-transposition events. Thus, while it is possible to identify excision-only events by changes in GFP expression, the vast majority of GFP-bright progeny represent re-integration events.
Remobilization of transposons resident in the genome may result in chromosomal rearrangements near the donor locus [34
]. In mice, germline remobilization of SB
transposons from a high-copy number (approximately 30 copies) concatemer resulted in frequent alteration of the genomic sequences flanking the transposon donor locus; nine out of nine remobilized pedigrees examined displayed genomic alterations spanning 105
bp to 107
bp flanking the donor site [41
]. To determine whether SB
remobilization in the frog resulted in similar genomic alterations near the donor locus, we examined the sequences flanking the 8F donor locus by PCR. Genomic DNA samples from eight remobilization events and eight excision-only events were used to amplify the sequences flanking the 5' and 3' ends of the 8F concatemer on chromosome 6. In each case, genomic PCR using primers that amplify the 5' and 3' junctions of the 8F locus generated the appropriate sized products (data not shown), indicating that the sequences directly flanking the donor locus are intact following excision of pT2
βGFP transposons from the donor site.
Sequence analysis of the re-integration target sites indicated a similar base distribution flanking the canonical TA dinucleotide to that observed with SB
integration in mammalian genomes [42
] (Figure ). Transposons of the Tc1/mariner
family, including SB
, integrate at TA dinucleotides. The consensus sequence for SB
integration in frogs, as in mammals, is a palindromic ATATA
TAT sequence, where the canonical TA target is in bold, although none of the re-integration events observed in the frog have this exact palindrome.
Cloning the integration sites of the novel loci indicated that the remobilized transposons frequently integrate near the parental locus (Figures and ; 12 out of 15 classed as local hopping, 80%). In two cases, we identified remobilization events that had re-integrated within the parental transposon concatemer on scaffold 57 (Table and Figure ). The scaffold identity was used to 'map' the chromosomal location [44
] of the novel integration events and showed that, while local hopping was more frequent, re-integration on other chromosomes was also detected (Figure ; three out of fifteen (20%) of integrations are on different chromosomes).
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 (more ...)
GFP-bright progeny from the outcross of 8F hopper frogs were raised to the adult stage, and outcrossed to demonstrate that the remobilized transposon alleles are stably transmitted through the germline. Genomic DNA was harvested from GFP-positive and GFP-negative siblings and used for Southern blot analysis and for cloning the novel integration site by EPTS LM-PCR. For example, remobilized female frog 62E3 produced GFP-bright progeny and integration site analysis showed a single copy of the pT2βGFP transposon on scaffold 140 (140:1237072). The novel re-integration event was on the same chromosome as the donor locus (chromosome 6, linkage group 2), approximately 1 cM from the parental 8F concatemer, and represents a local hop (data not shown). The 62E3 integration event was in the 3' UTR of a muscle-related coiled coil protein (GenBank accession number XM_002935280.1) gene.
The pT2βGFP 7M founder had a concatemer of 8 to 10 pT2βGFP transposons at a single locus within a repeat on scaffold 38 (Linkage Group 10, chromosome 10), and mapped, by fluorescence in situ hybridization (FISH) analysis, near a telomere on chromosome 10 (Figure ). Double transgenic 7M hopper frogs were generated by breeding heterozygous pT2βGFP 7M F1 frogs with heterozygous CAGGS-SB10;γcRFP 2M F1 frogs, and the progeny were sorted for GFP-positive and RFP-positive expression. The double-heterozygous 7M hopper frogs were outcrossed with wild-type animals and remobilization events were scored in the progeny by observing the outcross population for changes in GFP expression. To date, ten 7M hoppers have been outcrossed and 112 remobilized (GFP-bright) tadpoles have been identified from 11,646 GFP-positive progeny (Figure ). Genomic DNA from several GFP-bright tadpoles was analyzed by Southern blot, and this data verified that the banding pattern had changed from the parental 7M pattern, indicative of transposon remobilization. The novel integration sites were cloned (Table ), and sequence analysis confirmed that the remobilized transposons had re-integrated via canonical SB-mediated transposition (data not shown). The average apparent rate of remobilization was approximately 1%, and is five-times higher than that observed for the 8F hopper animals. The higher rate of remobilization observed in the 7M hoppers compared to the 8F hoppers may be due to the increased number of potential substrate transposons in the donor concatemer (three for 8F compared with 8 to 10 for 7M). A range of remobilization activities, from 0% to 5%, was noted between the different 7M hopper frogs. The 7M hoppers were produced by breeding frogs that were heterozygous for the SB10 enzyme transgene with frogs that were heterozygous for the pT2βGFP 7M allele. Double-heterozygous males (7Mhopper♂1, 7Mhopper♂2, 7Mhopper♂3, 7Mhopper♂5, 7Mhopper♂14, 7Mhopper♂20) produced offspring with an average remobilization frequency of approximately 0.44%. The frequency of GFP-positive progeny in the outcrosses from these males was approximately 50%, as expected for the Mendelian inheritance of a heterozygous dominant allele. Two 7M hopper male frogs (7Mhopper♂9 and 7Mhopper♂11) produced a much higher rate of remobilized (GFP-bright) tadpoles than their siblings (approximately 1.9% compared with 0.44% for male sibling hoppers). Intriguingly, these animals appear to be homozygous for both the enzyme (CAGGS-SB10;γcRFP) and substrate (pT2βGFP) transgenes; 100% of the outcross progeny were RFP-positive and nearly all (> 98%) were also GFP-positive. Southern blot analysis of the outcross progeny indicated that all of the GFP-positive animals (n = 107 for 7Mhopper♂9; n = 114 for 7Mhopper♂11) had inherited the 7M concatemer, and the banding pattern was identical to the parental locus. The GFP-bright tadpoles in the outcross populations of 7Mhopper♂9 and 7Mhopper♂11 showed changes in parental 7M locus indicative of excision and re-integration of a transposon from the substrate donor locus. The rare (< 2%) GFP-negative tadpoles observed in the outcross populations did not inherit the pT2βGFP transgene as determined by Southern blot and genomic PCR for GFP sequences (data not shown). The unexpected non-Mendelian inheritance of hoppers 7M♂9 and 7M♂11 is unexplained; however, this data suggests that increasing the copy number of the transposon substrates in the hopper lines may increase the remobilization frequency observed in the outcross population.
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 pT2 (more ...)
Integration site analysis for remobilized progeny from 7M Hoppers
To date, four 7M female hopper frogs have been outcrossed, and the mean remobilization rate from these animals is 2.54%. This may reflect individual differences in excision and reintegration activities between different hopper animals, or it may indicate that remobilization, driven by the CAGGS-SB10 transgene, is more efficient in the female germline (mean 2.54%; n = 4) than in the male germline (mean 0.76%; n = 9, unpaired Student's t-test, P = 0.0088, degrees of freedom, 11). With the 8F hoppers, we observed a modest increase in the mean remobilization efficiency with the female hoppers (0.56%; n = 2) compared to the male hoppers (0.25%; n = 7); however, due to the small sample size, this may not be statistically significant (Student's t-test P = 0.25, degrees of freedom, 1).
The 7M pT2βGFP concatemer is located on scaffold 38 that maps to chromosome 10 (Figure and ). Genomic DNA harvested from representative GFP-bright tadpoles from the 7M hopper outcrosses was analyzed by Southern blot and the novel integration sites were cloned by EPTS LM-PCR. As noted for the remobilized 8F hopper progeny above, the novel integration events from the 7M hoppers were canonical SB-mediated transposition events. As determined for the remobilization events from the 8F hopper frogs, a strong bias for local re-integration was observed for the 7M hoppers; re-integration events on the same scaffold (scaffold 38) as the transposon donor were cloned from the GFP-bright tadpoles.