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In vitro fertilized mouse eggs (C57BL/6N), followed by ultrarapid freezing were used for production of transgenic mice by microinjection of the chicken beta-actin promoter-driven the firefly luciferase cDNA (beta act-Luc). Following micromanipulation, the survival rates of the cryopreserved eggs and of the fresh in vitro fertilized eggs (control) were 70.8% (131/185) and 71.9% (159/221), respectively. After transferring them into oviducts of psudopregnant recipients on Day 1, 13.6% (17/125) of the cryopreserved eggs developed to live offspring and 14.1% (21/149) of fresh eggs did so. It was confirmed by Southern blotting analysis that each two transgenic mice were produced from the cryopreserved eggs (12%, 2/17) and the fresh eggs (10%, 2/21). All of transgenic mice produced from both eggs showed the expression of the luciferase gene. These results indicate that the in vitro fertilized eggs cryopreserved by ultrarapid freezing, can be, easily and conveniently, used for generation of transgenic mice.

We cryopreserved pronuclear stage-embryos from the Transgenic rats produced by introducing the rat GH antisense transgene, by ultrarapid freezing. The embryos were obtained by fertilization in vitro with the sperm of homozygous males for the transgene. After thawing of a part of cryopreserved embryos (97 embryos), the 40 embryos (41%) were morphologically normal, and were transferred to oviducts of recipients. All four live young (10%) obtained (one male and three females) preserved the transgene and exhibited dwarfism. We also produced the homozygous transgenic rats after brother-sister mating of those young. These results demonstrated the possibility of the cryopreservation of embryos by ultrarapid freezing for sustaining transgenic rat line.

Two transgenic mice lines were produced by introducing the rat GH antisense transgene and the chicken beta-actin promoter/firefly luciferase hybrid gene in our laboratory. We cryopreserved the transgenic embryos, obtained by fertilization in vitro with the sperm of hemizygous males for the transgene, by the ultrarapid freezing. The survival rates of cryopreserved 2-cell embryos were high (> 70%) at thawing in both lines and 53% and 16% of cryopreserved 2-cell embryos, respectively, developed to live young after transferring to oviducts of recipients. Each transgene was detected at about 40% of the live young from two transgenic lines. These results indicated that the cryopreservation of embryos by ultrarapid freezing was valuable for sustaining transgenic mouse lines without genetic contaminations.

Spermatozoa of a homozygous transgenic mouse, in which the firefly luciferase gene was expressed under the control of beta-actin promoter, were frozen at -196 degrees C. One fourth of the frozen sperm was later thawed and used for in vitro fertilization. Thirty-six of 65 oocytes (55.4%) developed to the 2-cell stage. All the 2-cell embryos were transferred to the oviducts of pseudopregnant recipients and 23 young (63.9%, 23/36) were born. All of young analyzed carried the transgene and showed the luciferase gene expression.