Reproductive Technology

Genomic DNA Damage in Mouse Transgenesis

Abstract
Creating transgenic mammals is currently a very low efficiency process. In addition to problems with transgene integration and unpredictable expression patterns of the inserted gene, embryo loss occurs at various developmental stages. In this study we demonstrated that this loss is due to chromosomal damage. We examined the integrity of chromosomes in embryos produced by pronuclear microinjection, intracytoplasmic sperm injection (ICSI) and in vitro fertilization (IVF)-mediated transgenesis, and correlated with the ability of embryos to develop in vitro and yield transgenic morula/blastocysts. Chromosome analysis after pronuclear microinjection was done for zygotes, and for parthenogenetic and androgenetic embryos. In all pronuclear injection groups a significant oocyte arrest and increased incidence of chromosome breaks were observed both after transgenic DNA and sham injection. This indicated that the DNA damage was a transgene-independent effect. In ICSI mediated transgenesis there was no significant oocyte arrest. The observed chromosome damage was lower than in the zygote pronuclear microinjection, and was dependent on the presence of exogenous DNA. The occurrence of DNA breaks, measured by comet assay analysis of sperm prior to ICSI suggested that DNA damage was present in sperm before fertilization. Embryo development in vitro and transgene expression at the morula/blastocyst stage was higher in ICSI mediated transgenesis than after pronuclear microinjection in zygotes. Sperm mediated gene transfer via IVF did not affect chromosome integrity, allowed good embryo development but did not yield any transgenic embryos. This study demonstrated that DNA damage occurred in both pronuclear microinjection and ICSI-mediated transgenesis but through different mechanisms.

Key words: Embryo • Gamete Biology • Assisted Reproductive Technology • In vitro fertilization • Sperm