Transgenic Research

, Volume 16, Issue 3, pp 333–339

Active integration: new strategies for transgenesis

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

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

Transposon Transposase Site-specific Retrovirus Recombinase 

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Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Eric T. Shinohara
    • 1
  • Joseph M. Kaminski
    • 2
  • David J. Segal
    • 3
  • Pawel Pelczar
    • 4
  • Ravindra Kolhe
    • 2
  • Thomas Ryan
    • 5
  • Craig J. Coates
    • 6
  • Malcolm J. Fraser
    • 7
  • Alfred M. Handler
    • 8
  • Ryuzo Yanagimachi
    • 9
  • Stefan Moisyadi
    • 9
  1. 1.Department of Radiation Oncology, Abramson Cancer CenterUniversity of PennsylvaniaPhiladelphiaUSA
  2. 2.Medical College of Georgia Cancer CenterMolecular Chaperone/Radiobiology and Cancer VirologyAugustUSA
  3. 3.Department of PharmacologyUniversity of California Genome CenterDavisUSA
  4. 4.Institute of Laboratory and Animal SciencesUniversity of ZurichZurichSwitzerland
  5. 5.Department of Biochemistry and Molecular GeneticsUniversity of Alabama at BirminghamBirminghamUSA
  6. 6.Department of EntomologyTexas A&M UniversityCollege StationUSA
  7. 7.Department of Biological SciencesUniversity of Notre DameNotre DameUSA
  8. 8.Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research ServiceU.S. Department of AgricultureGainesvilleUSA
  9. 9.John A. Burns School of Medicine, University of Hawaii at ManoaHonoluluUSA

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