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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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Passport functions in cells from a wide variety of vertebrate sources. (A) A Passport transposon that expresses Puromycin phosphotransferase was co-transfected with a source of Passport transpoase, pKC-PTs1 (+PTs) or pCMV-Bgal (–PTs). Cells were selected in puromycin and stable colonies were counted. (B) HeLa, CHO, Vero and HT1080 cells displayed an increase in stable colony formation with the addition of Passport transposase. (C) 3T3, TT, DF1 and PEGE cells produced fewer colonies under these transfection conditions; however, the addition of Passport transposase significantly improved colony formation. The addition of transposase rather than beta-galactosidase significantly increased colony formation in all cell types (P < 0.05, except CHO where P = 0.07).
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Figure 1: Passport functions in cells from a wide variety of vertebrate sources. (A) A Passport transposon that expresses Puromycin phosphotransferase was co-transfected with a source of Passport transpoase, pKC-PTs1 (+PTs) or pCMV-Bgal (–PTs). Cells were selected in puromycin and stable colonies were counted. (B) HeLa, CHO, Vero and HT1080 cells displayed an increase in stable colony formation with the addition of Passport transposase. (C) 3T3, TT, DF1 and PEGE cells produced fewer colonies under these transfection conditions; however, the addition of Passport transposase significantly improved colony formation. The addition of transposase rather than beta-galactosidase significantly increased colony formation in all cell types (P < 0.05, except CHO where P = 0.07).

Mentions: The SB, Frog Prince and HsMar1 transposon systems are active in a wide array of vertebrate cells (11–13), although to differing degrees. In order to assess the ability and ubiquity of Passport function, we undertook an analysis of TnT in human (HeLa, HT1080), monkey (Vero), pig (PEGE), hamster (CHO), mouse (3T3), chicken (DF1) and turkey (TT) cells using a Passport transposon containing a puromycin thymidine kinase fusion protein (25) driven by the mouse PGK promoter (pPTnP-PTK). Cells were transfected with the pPTnP-PTK transposon and a Passport transposase expression construct (pKC-PTs) at a Tn:Ts molar ratio of 1:0.5, or with the molar equivalent of pCMV-βgal. Following transfection, replicates of ∼30 000 cells were plated and selected in puromycin, fixed, stained and enumerated. In all cases, Passport-dependent TnT resulted in the generation of a number of puromycin-resistant colonies far exceeding that observed for controls lacking transposase, in the case of HeLa cells reflecting at least a 40-fold enhancement (Figure 1). As with other transposon systems (12,26), TnT varied between cell types (as did background-resistant colony formation), although comparing relative transpositional activity across cell lines may be confounded by the fact that transfections were conducted under identical conditions that may be suboptimal for some cell lines. Nonetheless, native Passport is functional in cells from a broad sampling of vertebrate species.Figure 1.


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)

Passport functions in cells from a wide variety of vertebrate sources. (A) A Passport transposon that expresses Puromycin phosphotransferase was co-transfected with a source of Passport transpoase, pKC-PTs1 (+PTs) or pCMV-Bgal (–PTs). Cells were selected in puromycin and stable colonies were counted. (B) HeLa, CHO, Vero and HT1080 cells displayed an increase in stable colony formation with the addition of Passport transposase. (C) 3T3, TT, DF1 and PEGE cells produced fewer colonies under these transfection conditions; however, the addition of Passport transposase significantly improved colony formation. The addition of transposase rather than beta-galactosidase significantly increased colony formation in all cell types (P < 0.05, except CHO where P = 0.07).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 1: Passport functions in cells from a wide variety of vertebrate sources. (A) A Passport transposon that expresses Puromycin phosphotransferase was co-transfected with a source of Passport transpoase, pKC-PTs1 (+PTs) or pCMV-Bgal (–PTs). Cells were selected in puromycin and stable colonies were counted. (B) HeLa, CHO, Vero and HT1080 cells displayed an increase in stable colony formation with the addition of Passport transposase. (C) 3T3, TT, DF1 and PEGE cells produced fewer colonies under these transfection conditions; however, the addition of Passport transposase significantly improved colony formation. The addition of transposase rather than beta-galactosidase significantly increased colony formation in all cell types (P < 0.05, except CHO where P = 0.07).
Mentions: The SB, Frog Prince and HsMar1 transposon systems are active in a wide array of vertebrate cells (11–13), although to differing degrees. In order to assess the ability and ubiquity of Passport function, we undertook an analysis of TnT in human (HeLa, HT1080), monkey (Vero), pig (PEGE), hamster (CHO), mouse (3T3), chicken (DF1) and turkey (TT) cells using a Passport transposon containing a puromycin thymidine kinase fusion protein (25) driven by the mouse PGK promoter (pPTnP-PTK). Cells were transfected with the pPTnP-PTK transposon and a Passport transposase expression construct (pKC-PTs) at a Tn:Ts molar ratio of 1:0.5, or with the molar equivalent of pCMV-βgal. Following transfection, replicates of ∼30 000 cells were plated and selected in puromycin, fixed, stained and enumerated. In all cases, Passport-dependent TnT resulted in the generation of a number of puromycin-resistant colonies far exceeding that observed for controls lacking transposase, in the case of HeLa cells reflecting at least a 40-fold enhancement (Figure 1). As with other transposon systems (12,26), TnT varied between cell types (as did background-resistant colony formation), although comparing relative transpositional activity across cell lines may be confounded by the fact that transfections were conducted under identical conditions that may be suboptimal for some cell lines. Nonetheless, native Passport is functional in cells from a broad sampling of vertebrate species.Figure 1.

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