Maintenance of Transgenes in Rats: The Contributions of Embryo Cryopreservation and Intracytoplasmic Sperm/Spermatid Injection.

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The maintenance of transgenes in rats is reviewed with special references to embryo cryopreservation (transgene banking) and intracytoplasmic injection of spermatozoa or round spermatids (transgene passage). Production of transgenic rats by pronuclear DNA microinjection is easily performed, particularly using the micro-vibration method. Two-cell stage embryos derived from the mating of transgenic rats with wild-type rats can be successfully cryopreserved by conventional two-step freezing in the presence of 10% dimethylsulfoxide. However, the transmission rates of transgenes into newborn offspring are lower than the 50% which is expected in simple Mendelian inheritance. Furthermore, some transgenic rats are unable to transmit their transgenes to subsequent generations. When the inability of male transgenic rats to transmit the transgene is not associated with mosaicism, microinsemination techniques such as intracytoplasmic sperm injection (ICSI) and round spermatid injection (ROSI) can be used for passage of the transgenes. Our ICSI procedure includes the use of a piezo-driven small-sized injection pipette (2-4 μm in diameter), isolated sperm heads, and freshly ovulated oocytes. In the case of ROSI, round spermatids are injected into Sr2+-activated oocytes using a piezo-driven middle-sized pipette (5-6 μm in diameter). These microinsemination techniques can be combined with cryopreservation of sperm and spermatid cells, leading to successful transmission of transgenes carried in male gametes.

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