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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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Examination of overproduction inhibition. (A) To examine the effect of transposase dose on transposition rates, a constant amount of pTnP-GeN (75 fmol) was co-transfected with five different molar ratios of transposase expression vector driven by either the human UbC promoter (pKUb-Ts) or the mCAGs promoter (pKC-Ts), where T and Ts generically refer to either SB or Passport components. In all cases the total amount of DNA transfected was adjusted to 2 µg by the addition of the appropriate amount of pCMV-Bgal. After transfection and selection in G418, colonies were counted and the data compared to an internal reference transfection of SB at a ratio of 1:1U. The raw data for the internal reference transfection came from a total of 30 replicates and ranged from 68 to 324, with a median of 150 and a mean of 170 (data not shown). The relative transposition efficiencies confirm overproduction inhibition of (B) the SB transposon system and (C) demonstrate overproduction inhibition of the Passport transposon system. Error bars represent the SE.
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Figure 2: Examination of overproduction inhibition. (A) To examine the effect of transposase dose on transposition rates, a constant amount of pTnP-GeN (75 fmol) was co-transfected with five different molar ratios of transposase expression vector driven by either the human UbC promoter (pKUb-Ts) or the mCAGs promoter (pKC-Ts), where T and Ts generically refer to either SB or Passport components. In all cases the total amount of DNA transfected was adjusted to 2 µg by the addition of the appropriate amount of pCMV-Bgal. After transfection and selection in G418, colonies were counted and the data compared to an internal reference transfection of SB at a ratio of 1:1U. The raw data for the internal reference transfection came from a total of 30 replicates and ranged from 68 to 324, with a median of 150 and a mean of 170 (data not shown). The relative transposition efficiencies confirm overproduction inhibition of (B) the SB transposon system and (C) demonstrate overproduction inhibition of the Passport transposon system. Error bars represent the SE.

Mentions: Overproduction inhibition, where excess transposase reduces the rate of transposition, is a hallmark of Tc1/mariner elements (5). We thus undertook an analysis of this effect for Passport and compared its sensitivity to that of the well-characterized SB transposon system (27,28). A series of transfections was performed with varying ratios of transposase to transposon vector to measure the effect of increasing transposase concentration on the rate of transposition. In addition, two promoters [human UbC (29) and mCAG (22,30)] were used to drive expression of the Passport transposase across a broad range of transposase levels (Figure 2). In HT1080 cells, gene expression from the mCAGs promoter is 5- to 10-fold higher than from the UbC promoter (data not shown). A constant amount of transposon (pPTnP-GeN, 75 fmol) was co-transfected with transposase vector containing either the UbC or mCAGs promoter (pKC-PTs or pKUb-PTs at a Tn:Ts molar ratio of 1:0.2, 1:0.5, 1:1, 1:2 or 1:5 corresponding to 15, 37.5, 75, 150 and 375 fmol of transposase plasmid). The total amount of transfected DNA was kept at 2 µg by supplementing with pCMV-βgal DNA. To compare to the SB transposon system, identical reactions were performed with an SB transposon (pKT2P-GeN) and SB11 transposase expressed from the UbC and mCAGs promoters (pKUb-SB11 and pKC-SB11). Following transfection, two replicates of ∼30 000 cells were plated and selected in G418 for 10–14 days, fixed, stained and the resulting colonies enumerated. Our previous studies indicated that a molar ratio of 1:1 SB transposon to SB transposase expressed from the human UbC promoter resulted in near-optimal transposition rates for the SB transposon system. Therefore to correct for any variation in transfection or selection, a 1:1 ratio of pKT2P-GeN:pKUb-SB11 was included as in internal standard for each day of transfection. The relative sensitivity of the two transposon systems to overproduction inhibition is presented in Figure 2B and C, where colony formation is expressed relative to the contemporary pKT2P-GeN:pKUb-SB11 internal standard. As shown in Figure 2B, the hyperactive SB system resulted in more than twice as many colonies as the native Passport system (Figure 2C) at their respective optimal Tn:Ts ratios. As expected, the SB transposon system is sensitive to overproduction inhibition. The peak transpositional activity for Passport was observed using a 1:5 ratio of pPTnP-GeN:pKUb-PTs1 or a 1:0.2 ratio of pPTnP-GeN:pKC-PTs1, beyond which increasing transposase expression resulted in reduced transposition, indicating that Passport is indeed susceptible to overproduction inhibition.Figure 2.


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)

Examination of overproduction inhibition. (A) To examine the effect of transposase dose on transposition rates, a constant amount of pTnP-GeN (75 fmol) was co-transfected with five different molar ratios of transposase expression vector driven by either the human UbC promoter (pKUb-Ts) or the mCAGs promoter (pKC-Ts), where T and Ts generically refer to either SB or Passport components. In all cases the total amount of DNA transfected was adjusted to 2 µg by the addition of the appropriate amount of pCMV-Bgal. After transfection and selection in G418, colonies were counted and the data compared to an internal reference transfection of SB at a ratio of 1:1U. The raw data for the internal reference transfection came from a total of 30 replicates and ranged from 68 to 324, with a median of 150 and a mean of 170 (data not shown). The relative transposition efficiencies confirm overproduction inhibition of (B) the SB transposon system and (C) demonstrate overproduction inhibition of the Passport transposon system. Error bars represent the SE.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: Examination of overproduction inhibition. (A) To examine the effect of transposase dose on transposition rates, a constant amount of pTnP-GeN (75 fmol) was co-transfected with five different molar ratios of transposase expression vector driven by either the human UbC promoter (pKUb-Ts) or the mCAGs promoter (pKC-Ts), where T and Ts generically refer to either SB or Passport components. In all cases the total amount of DNA transfected was adjusted to 2 µg by the addition of the appropriate amount of pCMV-Bgal. After transfection and selection in G418, colonies were counted and the data compared to an internal reference transfection of SB at a ratio of 1:1U. The raw data for the internal reference transfection came from a total of 30 replicates and ranged from 68 to 324, with a median of 150 and a mean of 170 (data not shown). The relative transposition efficiencies confirm overproduction inhibition of (B) the SB transposon system and (C) demonstrate overproduction inhibition of the Passport transposon system. Error bars represent the SE.
Mentions: Overproduction inhibition, where excess transposase reduces the rate of transposition, is a hallmark of Tc1/mariner elements (5). We thus undertook an analysis of this effect for Passport and compared its sensitivity to that of the well-characterized SB transposon system (27,28). A series of transfections was performed with varying ratios of transposase to transposon vector to measure the effect of increasing transposase concentration on the rate of transposition. In addition, two promoters [human UbC (29) and mCAG (22,30)] were used to drive expression of the Passport transposase across a broad range of transposase levels (Figure 2). In HT1080 cells, gene expression from the mCAGs promoter is 5- to 10-fold higher than from the UbC promoter (data not shown). A constant amount of transposon (pPTnP-GeN, 75 fmol) was co-transfected with transposase vector containing either the UbC or mCAGs promoter (pKC-PTs or pKUb-PTs at a Tn:Ts molar ratio of 1:0.2, 1:0.5, 1:1, 1:2 or 1:5 corresponding to 15, 37.5, 75, 150 and 375 fmol of transposase plasmid). The total amount of transfected DNA was kept at 2 µg by supplementing with pCMV-βgal DNA. To compare to the SB transposon system, identical reactions were performed with an SB transposon (pKT2P-GeN) and SB11 transposase expressed from the UbC and mCAGs promoters (pKUb-SB11 and pKC-SB11). Following transfection, two replicates of ∼30 000 cells were plated and selected in G418 for 10–14 days, fixed, stained and the resulting colonies enumerated. Our previous studies indicated that a molar ratio of 1:1 SB transposon to SB transposase expressed from the human UbC promoter resulted in near-optimal transposition rates for the SB transposon system. Therefore to correct for any variation in transfection or selection, a 1:1 ratio of pKT2P-GeN:pKUb-SB11 was included as in internal standard for each day of transfection. The relative sensitivity of the two transposon systems to overproduction inhibition is presented in Figure 2B and C, where colony formation is expressed relative to the contemporary pKT2P-GeN:pKUb-SB11 internal standard. As shown in Figure 2B, the hyperactive SB system resulted in more than twice as many colonies as the native Passport system (Figure 2C) at their respective optimal Tn:Ts ratios. As expected, the SB transposon system is sensitive to overproduction inhibition. The peak transpositional activity for Passport was observed using a 1:5 ratio of pPTnP-GeN:pKUb-PTs1 or a 1:0.2 ratio of pPTnP-GeN:pKC-PTs1, beyond which increasing transposase expression resulted in reduced transposition, indicating that Passport is indeed susceptible to overproduction inhibition.Figure 2.

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