Active integration

New strategies for transgenesis

Eric T. Shinohara, Joseph Michael Kaminski, David J. Segal, Pawel Pelczar, Ravindra Bharat Kolhe, Thomas Ryan, Craig J. Coates, Malcolm J. Fraser, Alfred M. Handler, Ryuzo Yanagimachi, Stefan Moisyadi

Research output: Contribution to journalReview article

30 Citations (Scopus)

Abstract

This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

Original languageEnglish (US)
Pages (from-to)333-339
Number of pages7
JournalTransgenic Research
Volume16
Issue number3
DOIs
StatePublished - Jun 1 2007

Fingerprint

Gene Transfer Techniques
Transposases
Transgenes
Genetically Modified Animals
Intracytoplasmic Sperm Injections
intracytoplasmic sperm injection
transgenic animals
transgenes
Genome
Genetic Therapy
Oocytes
Transfection
methodology
gene therapy
RNA
transfection
Recombinases
Mosaicism
DNA
Microinjections

Keywords

  • Recombinase
  • Retrovirus
  • Site-specific
  • Transposase
  • Transposon

ASJC Scopus subject areas

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

Cite this

Shinohara, E. T., Kaminski, J. M., Segal, D. J., Pelczar, P., Kolhe, R. B., Ryan, T., ... Moisyadi, S. (2007). Active integration: New strategies for transgenesis. Transgenic Research, 16(3), 333-339. https://doi.org/10.1007/s11248-007-9077-z

Active integration : New strategies for transgenesis. / Shinohara, Eric T.; Kaminski, Joseph Michael; Segal, David J.; Pelczar, Pawel; Kolhe, Ravindra Bharat; Ryan, Thomas; Coates, Craig J.; Fraser, Malcolm J.; Handler, Alfred M.; Yanagimachi, Ryuzo; Moisyadi, Stefan.

In: Transgenic Research, Vol. 16, No. 3, 01.06.2007, p. 333-339.

Research output: Contribution to journalReview article

Shinohara, ET, Kaminski, JM, Segal, DJ, Pelczar, P, Kolhe, RB, Ryan, T, Coates, CJ, Fraser, MJ, Handler, AM, Yanagimachi, R & Moisyadi, S 2007, 'Active integration: New strategies for transgenesis', Transgenic Research, vol. 16, no. 3, pp. 333-339. https://doi.org/10.1007/s11248-007-9077-z
Shinohara ET, Kaminski JM, Segal DJ, Pelczar P, Kolhe RB, Ryan T et al. Active integration: New strategies for transgenesis. Transgenic Research. 2007 Jun 1;16(3):333-339. https://doi.org/10.1007/s11248-007-9077-z
Shinohara, Eric T. ; Kaminski, Joseph Michael ; Segal, David J. ; Pelczar, Pawel ; Kolhe, Ravindra Bharat ; Ryan, Thomas ; Coates, Craig J. ; Fraser, Malcolm J. ; Handler, Alfred M. ; Yanagimachi, Ryuzo ; Moisyadi, Stefan. / Active integration : New strategies for transgenesis. In: Transgenic Research. 2007 ; Vol. 16, No. 3. pp. 333-339.
@article{b80b2b545bba4b8388cb29fda9975dc9,
title = "Active integration: New strategies for transgenesis",
abstract = "This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45{\%} of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed {"}Active Transgenesis{"} to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.",
keywords = "Recombinase, Retrovirus, Site-specific, Transposase, Transposon",
author = "Shinohara, {Eric T.} and Kaminski, {Joseph Michael} and Segal, {David J.} and Pawel Pelczar and Kolhe, {Ravindra Bharat} and Thomas Ryan and Coates, {Craig J.} and Fraser, {Malcolm J.} and Handler, {Alfred M.} and Ryuzo Yanagimachi and Stefan Moisyadi",
year = "2007",
month = "6",
day = "1",
doi = "10.1007/s11248-007-9077-z",
language = "English (US)",
volume = "16",
pages = "333--339",
journal = "Transgenic Research",
issn = "0962-8819",
publisher = "Springer Netherlands",
number = "3",

}

TY - JOUR

T1 - Active integration

T2 - New strategies for transgenesis

AU - Shinohara, Eric T.

AU - Kaminski, Joseph Michael

AU - Segal, David J.

AU - Pelczar, Pawel

AU - Kolhe, Ravindra Bharat

AU - Ryan, Thomas

AU - Coates, Craig J.

AU - Fraser, Malcolm J.

AU - Handler, Alfred M.

AU - Yanagimachi, Ryuzo

AU - Moisyadi, Stefan

PY - 2007/6/1

Y1 - 2007/6/1

N2 - This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

AB - This paper presents novel methods for producing transgenic animals, with a further emphasis on how these techniques may someday be applied in gene therapy. There are several passive methods for transgenesis, such as pronuclear microinjection (PNI) and Intracytoplasmic Sperm Injection-Mediated Transgenesis (ICSI-Tr), which rely on the repair mechanisms of the host for transgene (tg) insertion. ICSI-Tr has been shown to be an effective means of creating transgenic animals with a transfection efficiency of approximately 45% of animals born. Furthermore, because this involves the injection of the transgene into the cytoplasm of oocytes during fertilization, limited mosaicism has traditionally occurred using this technique. Current active transgenesis techniques involve the use of viruses, such as disarmed retroviruses which can insert genes into the host genome. However, these methods are limited by the size of the sequence that can be inserted, high embryo mortality, and randomness of insertion. A novel active method has been developed which combines ICSI-Tr with recombinases or transposases to increase transfection efficiency. This technique has been termed "Active Transgenesis" to imply that the tg is inserted into the host genome by enzymes supplied into the oocyte during tg introduction. DNA based methods alleviate many of the costs and time associated with purifying enzyme. Further studies have shown that RNA can be used for the transposase source. Using RNA may prevent problems with continued transposase activity that can occur if a DNA transposase is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in mammalian systems and as further studies are done to elucidate modifications which improve piggyBac's specificity and efficacy, efficiency in creating transgenic animals should improve further. Subsequently, these methods may someday be used for gene therapy in humans.

KW - Recombinase

KW - Retrovirus

KW - Site-specific

KW - Transposase

KW - Transposon

UR - http://www.scopus.com/inward/record.url?scp=34247348957&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=34247348957&partnerID=8YFLogxK

U2 - 10.1007/s11248-007-9077-z

DO - 10.1007/s11248-007-9077-z

M3 - Review article

VL - 16

SP - 333

EP - 339

JO - Transgenic Research

JF - Transgenic Research

SN - 0962-8819

IS - 3

ER -