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.
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Department of Radiation Oncology, Abramson Cancer Center, University of Pennsylvania, Philadelphia, PA, 19104, USA
Eric T. Shinohara
Medical College of Georgia Cancer Center, Molecular Chaperone/Radiobiology and Cancer Virology, August, GA, 30912, USA
Joseph M. Kaminski & Ravindra Kolhe
Department of Pharmacology, University of California Genome Center, Davis, CA, 95616, USA
David J. Segal
Institute of Laboratory and Animal Sciences, University of Zurich, Zurich, Switzerland
Pawel Pelczar
Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, Birmingham, AL, 35294, USA
Thomas Ryan
Department of Entomology, Texas A&M University, College Station, TX, 77843, USA
Craig J. Coates
Department of Biological Sciences, University of Notre Dame, Notre Dame, IN, 46556, USA
Malcolm J. Fraser
Center for Medical, Agricultural, and Veterinary Entomology, Agricultural Research Service, U.S. Department of Agriculture, 1700 Southwest 23rd Drive, Gainesville, FL, 32608, USA
Alfred M. Handler
John A. Burns School of Medicine, University of Hawaii at Manoa, IBR E-108, 1960 East-West Road, Honolulu, HI, 96813, USA
Ryuzo Yanagimachi & Stefan Moisyadi
Correspondence to Joseph M. Kaminski or Stefan Moisyadi.
About this article Cite this articleShinohara, E.T., Kaminski, J.M., Segal, D.J. et al. Active integration: new strategies for transgenesis. Transgenic Res 16, 333–339 (2007). https://doi.org/10.1007/s11248-007-9077-z
Accepted: 27 January 2007
Published: 06 March 2007
Issue Date: June 2007
DOI: https://doi.org/10.1007/s11248-007-9077-z
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