|Handler, Alfred - Al|
Submitted to: Transgenic Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/27/2007
Publication Date: 3/6/2007
Citation: Shinohara, E.T., Kaminski, J.M., Segal, D.J., Pelczar, P., Kolhe, R., Ryan, T., Coates, C.J., Fraser, M.J., Handler, A.M., Yanagimachi, R., Moisyadi, S. 2007. Active integration: New strategies for transgenesis. Transgenic Research. 16:333-339. Interpretive Summary: The creation of transgenic strains of economically important insects for the development of more effective biological control programs is a major goal of our laboratory at CMAVE. Development of this methodology and strategies to effectively and safely utilize transgenic insects for biological control will depend upon new efficient strategies for integrating transgenes into insect host genomes. This paper describes a new system termed “Active Transgenesis” whereby the transposase enzyme and genes of interest reside within a single plasmid, versus a typical two-plasmid helper:vector system. RNA can also be used for transposase activity, which may prevent potential transgene instability if transposase DNA is integrated into the host genome. At present piggyBac is the most effective transposon for stable integration in insect and mammalian systems and as further studies are done to elucidate modifications which improve piggyBac’s specificity and efficacy, efficiency in creating transgenic organisms should improve further.
Technical 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 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 chimerism has traditionally occurred using this technique. Current active trangenesis 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 transposase activity. 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 models may someday be used for gene therapy in humans.