Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

Daniel F. Carlson, Aron M. Geurts, John R. Garbe, Chang Won Park, Artur Rangel-Filho, Scott M. O'Grady, Howard J. Jacob, Clifford J. Steer, David A. Largaespada, Scott C. Fahrenkrug

Research output: Contribution to journalArticle

23 Citations (Scopus)

Abstract

Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

Original languageEnglish (US)
Pages (from-to)29-45
Number of pages17
JournalTransgenic Research
Volume20
Issue number1
DOIs
StatePublished - 2011
Externally publishedYes

Fingerprint

Beauty
Gene Transfer Techniques
transposition (genetics)
Transgenes
transgenes
germ cells
transposons
methylation
Methylation
genetically modified organisms
mice
Transgenic Rats
cytosine
Cytosine
guanine
rats
Guanine
human diseases
Transgenic Mice
plasmids

Keywords

  • Methylation
  • Mouse
  • Rat
  • Sleeping Beauty
  • Transgenesis
  • Transposon

ASJC Scopus subject areas

  • Biotechnology
  • Genetics
  • Agronomy and Crop Science
  • Animal Science and Zoology

Cite this

Carlson, D. F., Geurts, A. M., Garbe, J. R., Park, C. W., Rangel-Filho, A., O'Grady, S. M., ... Fahrenkrug, S. C. (2011). Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Research, 20(1), 29-45. https://doi.org/10.1007/s11248-010-9386-5

Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. / Carlson, Daniel F.; Geurts, Aron M.; Garbe, John R.; Park, Chang Won; Rangel-Filho, Artur; O'Grady, Scott M.; Jacob, Howard J.; Steer, Clifford J.; Largaespada, David A.; Fahrenkrug, Scott C.

In: Transgenic Research, Vol. 20, No. 1, 2011, p. 29-45.

Research output: Contribution to journalArticle

Carlson, DF, Geurts, AM, Garbe, JR, Park, CW, Rangel-Filho, A, O'Grady, SM, Jacob, HJ, Steer, CJ, Largaespada, DA & Fahrenkrug, SC 2011, 'Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition', Transgenic Research, vol. 20, no. 1, pp. 29-45. https://doi.org/10.1007/s11248-010-9386-5
Carlson DF, Geurts AM, Garbe JR, Park CW, Rangel-Filho A, O'Grady SM et al. Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. Transgenic Research. 2011;20(1):29-45. https://doi.org/10.1007/s11248-010-9386-5
Carlson, Daniel F. ; Geurts, Aron M. ; Garbe, John R. ; Park, Chang Won ; Rangel-Filho, Artur ; O'Grady, Scott M. ; Jacob, Howard J. ; Steer, Clifford J. ; Largaespada, David A. ; Fahrenkrug, Scott C. / Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition. In: Transgenic Research. 2011 ; Vol. 20, No. 1. pp. 29-45.
@article{60bc2ff1e4af4bbc8d235561ff51b2b8,
title = "Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition",
abstract = "Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.",
keywords = "Methylation, Mouse, Rat, Sleeping Beauty, Transgenesis, Transposon",
author = "Carlson, {Daniel F.} and Geurts, {Aron M.} and Garbe, {John R.} and Park, {Chang Won} and Artur Rangel-Filho and O'Grady, {Scott M.} and Jacob, {Howard J.} and Steer, {Clifford J.} and Largaespada, {David A.} and Fahrenkrug, {Scott C.}",
year = "2011",
doi = "10.1007/s11248-010-9386-5",
language = "English (US)",
volume = "20",
pages = "29--45",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer Netherlands",
number = "1",

}

TY - JOUR

T1 - Efficient mammalian germline transgenesis by cis-enhanced Sleeping Beauty transposition

AU - Carlson, Daniel F.

AU - Geurts, Aron M.

AU - Garbe, John R.

AU - Park, Chang Won

AU - Rangel-Filho, Artur

AU - O'Grady, Scott M.

AU - Jacob, Howard J.

AU - Steer, Clifford J.

AU - Largaespada, David A.

AU - Fahrenkrug, Scott C.

PY - 2011

Y1 - 2011

N2 - Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

AB - Heightened interest in relevant models for human disease increases the need for improved methods for germline transgenesis. We describe a significant improvement in the creation of transgenic laboratory mice and rats by chemical modification of Sleeping Beauty transposons. Germline transgenesis in mice and rats was significantly enhanced by in vitro cytosine-phosphodiester-guanine methylation of transposons prior to injection. Heritability of transgene alleles was also greater from founder mice generated with methylated versus non-methylated transposon. The artificial methylation was reprogrammed in the early embryo, leading to founders that express the transgenes. We also noted differences in transgene insertion number and structure (single-insert versus concatemer) based on the influence of methylation and plasmid conformation (linear versus supercoiled), with supercoiled substrate resulting in efficient transpositional transgenesis (TnT) with near elimination of concatemer insertion. Combined, these substrate modifications resulted in increases in both the frequency of transgenic founders and the number of transgenes per founder, significantly elevating the number of potential transgenic lines. Given its simplicity, versatility and high efficiency, TnT with enhanced Sleeping Beauty components represents a compelling non-viral approach to modifying the mammalian germline.

KW - Methylation

KW - Mouse

KW - Rat

KW - Sleeping Beauty

KW - Transgenesis

KW - Transposon

UR - http://www.scopus.com/inward/record.url?scp=78651398529&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=78651398529&partnerID=8YFLogxK

U2 - 10.1007/s11248-010-9386-5

DO - 10.1007/s11248-010-9386-5

M3 - Article

C2 - 20352328

AN - SCOPUS:78651398529

VL - 20

SP - 29

EP - 45

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 1

ER -