Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells

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Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells. / Dalsgaard, Trine; Moldt, Brian; Sharma, Nynne; Wolf, Gernot; Schmitz, Alexander; Pedersen, Finn S; Mikkelsen, Jacob G.

I: Molecular Therapy, Bind 17, Nr. 1, 2009, s. 121-130.

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Dalsgaard, Trine ; Moldt, Brian ; Sharma, Nynne ; Wolf, Gernot ; Schmitz, Alexander ; Pedersen, Finn S ; Mikkelsen, Jacob G. / Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells. I: Molecular Therapy. 2009 ; Bind 17, Nr. 1. s. 121-130.

Bibtex

@article{15b97c50bd4a11dd99a9000ea68e967b,
title = "Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells",
abstract = "The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.Molecular Therapy (2008); doi:10.1038/mt.2008.224.",
author = "Trine Dalsgaard and Brian Moldt and Nynne Sharma and Gernot Wolf and Alexander Schmitz and Pedersen, {Finn S} and Mikkelsen, {Jacob G}",
year = "2009",
doi = "10.1038/mt.2008.224",
language = "English",
volume = "17",
pages = "121--130",
journal = "Molecular Therapy",
issn = "1525-0016",
publisher = "Nature Publishing Group",
number = "1",

}

RIS

TY - JOUR

T1 - Shielding of Sleeping Beauty DNA Transposon-delivered Transgene Cassettes by Heterologous Insulators in Early Embryonal Cells

AU - Dalsgaard, Trine

AU - Moldt, Brian

AU - Sharma, Nynne

AU - Wolf, Gernot

AU - Schmitz, Alexander

AU - Pedersen, Finn S

AU - Mikkelsen, Jacob G

PY - 2009

Y1 - 2009

N2 - The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.Molecular Therapy (2008); doi:10.1038/mt.2008.224.

AB - The Sleeping Beauty (SB) transposon system represents an important alternative to viral integrating vector systems but may, as its viral counterparts, be subject to transcriptional silencing. To investigate shielding of SB-delivered transgene cassettes against transcriptional repression, we establish silencing assays in which SB vector-containing F9 murine teratocarcinoma cell clones are identified by strategies that include or exclude selection for transgene expression. Among clones carrying one or more SB transposon vectors, more than one-third are immediately silenced, and most of the remaining clones move toward silencing during prolonged passage. In line with the lack of an intrinsic ability of SB to resist silencing, we show that the stable transfection rate of SB vectors in F9 cells is significantly improved by flanking the transgene with heterologous 5'-HS4 chicken beta-globin (cHS4) insulators. In approaches based on drug selection and subsequent flow-cytometric detection of transgene expression, clones containing cHS4-insulated vectors are to a much higher degree protected against transcriptional silencing, resulting in long-term expression of the fluorescent marker. Our findings demonstrate that SB vectors, prone for transcriptional silencing by positional effects in F9 cells, are protected by insulators. We believe that insulated SB-derived vectors will become useful tools in transposon-based transgenesis and therapeutic gene transfer.Molecular Therapy (2008); doi:10.1038/mt.2008.224.

U2 - 10.1038/mt.2008.224

DO - 10.1038/mt.2008.224

M3 - Journal article

C2 - 18985029

VL - 17

SP - 121

EP - 130

JO - Molecular Therapy

JF - Molecular Therapy

SN - 1525-0016

IS - 1

ER -