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Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells.

Clark KJ, Carlson DF, Leaver MJ, Foster LK, Fahrenkrug SC - Nucleic Acids Res. (2009)

Bottom Line: We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells.Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active.The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA.

ABSTRACT
The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

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Evaluation of diversity and number of Passport genomic integrations. (A) Transposase-mediated recombination into the genome should result in transposon fragments of variable length after digestion with AseI. The sizes of the fragments are dependent on the proximity of AseI recognition sites in the neighboring chromatin, and can be observed following Southern hybridization. (B) Commonly, when DNA integrates without the enzymatic activity of transposase, head-to-tail concatemers of variable length are formed and integrate into the genome by non-homologous end-joining. In this case, the size of this internal high-representative fragment (∼5.1 kb) is predictable based on the location of AseI sites within the transposon donor plasmid. (C) An image of our Southern hybridization of 15 independent HT1080 clones is shown. The paired head-to-tail arrows indicate the expected position of pPTnP-PTK concatemers that could potentially form during integration by non-homologous end joining. The line with outward facing arrowheads represents the size of the transposon and therefore the minimal expected size of a hybridizing fragment integrated by TnT. The asterisks mark two bands present in the HT1080 DNA that hybridize weakly with the PTK probe used here.
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Figure 3: Evaluation of diversity and number of Passport genomic integrations. (A) Transposase-mediated recombination into the genome should result in transposon fragments of variable length after digestion with AseI. The sizes of the fragments are dependent on the proximity of AseI recognition sites in the neighboring chromatin, and can be observed following Southern hybridization. (B) Commonly, when DNA integrates without the enzymatic activity of transposase, head-to-tail concatemers of variable length are formed and integrate into the genome by non-homologous end-joining. In this case, the size of this internal high-representative fragment (∼5.1 kb) is predictable based on the location of AseI sites within the transposon donor plasmid. (C) An image of our Southern hybridization of 15 independent HT1080 clones is shown. The paired head-to-tail arrows indicate the expected position of pPTnP-PTK concatemers that could potentially form during integration by non-homologous end joining. The line with outward facing arrowheads represents the size of the transposon and therefore the minimal expected size of a hybridizing fragment integrated by TnT. The asterisks mark two bands present in the HT1080 DNA that hybridize weakly with the PTK probe used here.

Mentions: To validate transposition we examined the number of integration events per cellular clone by Southern analysis. Transposition is supported by hybridizing fragments of varying lengths, corresponding to genomic restriction sites at varying distances from the transposon insertion-sites (Figure 3A). Non-transpositional DNA integration results in the formation of multi-copy concatemers (31) that are expected to result in a predictable restriction enzyme fragment derived from sites within the transposon vector (Figure 3B). The Southern analysis of DNA isolated from 15 HT1080 clones revealed that Passport indeed had transposed into the human genome, with one to four integrations per cellular clone (Figure 3C). Clones 4, 5 and 9 also contain a hybridizing band near the predicted size of a concatemer, although low signal intensity suggests low copy inserts not inconsistent with transposition.Figure 3.


Passport, a native Tc1 transposon from flatfish, is functionally active in vertebrate cells.

Clark KJ, Carlson DF, Leaver MJ, Foster LK, Fahrenkrug SC - Nucleic Acids Res. (2009)

Evaluation of diversity and number of Passport genomic integrations. (A) Transposase-mediated recombination into the genome should result in transposon fragments of variable length after digestion with AseI. The sizes of the fragments are dependent on the proximity of AseI recognition sites in the neighboring chromatin, and can be observed following Southern hybridization. (B) Commonly, when DNA integrates without the enzymatic activity of transposase, head-to-tail concatemers of variable length are formed and integrate into the genome by non-homologous end-joining. In this case, the size of this internal high-representative fragment (∼5.1 kb) is predictable based on the location of AseI sites within the transposon donor plasmid. (C) An image of our Southern hybridization of 15 independent HT1080 clones is shown. The paired head-to-tail arrows indicate the expected position of pPTnP-PTK concatemers that could potentially form during integration by non-homologous end joining. The line with outward facing arrowheads represents the size of the transposon and therefore the minimal expected size of a hybridizing fragment integrated by TnT. The asterisks mark two bands present in the HT1080 DNA that hybridize weakly with the PTK probe used here.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2651795&req=5

Figure 3: Evaluation of diversity and number of Passport genomic integrations. (A) Transposase-mediated recombination into the genome should result in transposon fragments of variable length after digestion with AseI. The sizes of the fragments are dependent on the proximity of AseI recognition sites in the neighboring chromatin, and can be observed following Southern hybridization. (B) Commonly, when DNA integrates without the enzymatic activity of transposase, head-to-tail concatemers of variable length are formed and integrate into the genome by non-homologous end-joining. In this case, the size of this internal high-representative fragment (∼5.1 kb) is predictable based on the location of AseI sites within the transposon donor plasmid. (C) An image of our Southern hybridization of 15 independent HT1080 clones is shown. The paired head-to-tail arrows indicate the expected position of pPTnP-PTK concatemers that could potentially form during integration by non-homologous end joining. The line with outward facing arrowheads represents the size of the transposon and therefore the minimal expected size of a hybridizing fragment integrated by TnT. The asterisks mark two bands present in the HT1080 DNA that hybridize weakly with the PTK probe used here.
Mentions: To validate transposition we examined the number of integration events per cellular clone by Southern analysis. Transposition is supported by hybridizing fragments of varying lengths, corresponding to genomic restriction sites at varying distances from the transposon insertion-sites (Figure 3A). Non-transpositional DNA integration results in the formation of multi-copy concatemers (31) that are expected to result in a predictable restriction enzyme fragment derived from sites within the transposon vector (Figure 3B). The Southern analysis of DNA isolated from 15 HT1080 clones revealed that Passport indeed had transposed into the human genome, with one to four integrations per cellular clone (Figure 3C). Clones 4, 5 and 9 also contain a hybridizing band near the predicted size of a concatemer, although low signal intensity suggests low copy inserts not inconsistent with transposition.Figure 3.

Bottom Line: We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells.Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active.The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

View Article: PubMed Central - PubMed

Affiliation: Department of Animal Science, University of Minnesota, St Paul, MN 55108, USA.

ABSTRACT
The Tc1/mariner family of DNA transposons is widespread across fungal, plant and animal kingdoms, and thought to contribute to the evolution of their host genomes. To date, an active Tc1 transposon has not been identified within the native genome of a vertebrate. We demonstrate that Passport, a native transposon isolated from a fish (Pleuronectes platessa), is active in a variety of vertebrate cells. In transposition assays, we found that the Passport transposon system improved stable cellular transgenesis by 40-fold, has an apparent preference for insertion into genes, and is subject to overproduction inhibition like other Tc1 elements. Passport represents the first vertebrate Tc1 element described as both natively intact and functionally active, and given its restricted phylogenetic distribution, may be contemporaneously active. The Passport transposon system thus complements the available genetic tools for the manipulation of vertebrate genomes, and may provide a unique system for studying the infiltration of vertebrate genomes by Tc1 elements.

Show MeSH
Related in: MedlinePlus