317 FACTORS AFFECTING PRODUCTION EFFICIENCY OF TRANSGENIC RATS BY ICSI-MEDIATED DNA TRANSFER

Abstract

Successful DNA transfer via intracytoplasmic sperm injection (ICSI) was first reported in mice (Perry et al. 1999, Science 284, 1180), and was recently extended to rats (Kato et al. 2004, Mol. Reprod. Dev. 69, 153). In the present study, factors affecting the production efficiency of transgenic rats by the ICSI-mediated DNA transfer were investigated. Cauda epididymal spermatozoa from Sprague-Dawley rats were sonicated (SO) and/or frozen-thawed (FT) for tail-cutting and membrane-disrupting. The sperm heads were exposed for 1 min to different concentrations (0.02–2.5 ng/μL) of 3.0 kb EGFP DNA solution, and then microinjected into denuded F1 (Donryu × LEW) rat oocytes. The optimal concentration of EGFP DNA was 0.1 ng/μL, as determined by the in vitro developmental competence into morulae/blastocysts and the EGFP expression of the ICSI oocytes. The presumptive 1- or 2-cell stage zygotes were transferred into oviducts of pseudopregnant Wistar females, and the presence of EGFP DNA in the offspring was examined by fluorescence under the 480 nm UV light. The production efficiency of transgenic rat offspring was 2.8% (2/71 zygotes transferred), 1.6% (1/63), and 3.3% (2/61) in the oocytes into which SO-, FT-, and SO+ FT-treated sperm heads were injected, respectively. The founder transgenic rats carrying EGFP DNA transmitted the transgenes to their progeny according to the Mendelian fashion (43.8–54.8%), suggesting the stable incorporation of the transgenes into rat genomes. Four rat strains (F344, LEW, Donryu, and Sprague-Dawley) were compared for their suitability as sperm/oocyte donors in the production of transgenic rats by ICSI with SO + FT-treated and 0.1 ng/μL EGFP DNA-exposed sperm heads. The production efficiency of the transgenic rats in the Sprague-Dawley strain (8.2%, 8/98) was significantly higher than that in LEW strain (0.9%, 1/114), while those in F344 (4.3%, 4/92) and Donryu (4.4%, 5/114) strains were intermediate. Attempts were made to introduce three other DNA constructs (5.0 kb plasmid and 208 kb BAC, both with Fyn gene, and 186 kb BAC with Svet1/IRES-Cre gene) into rat genomes by ICSI with SO + FT-treated and 0.1 ng/μL DNA-exposed sperm heads. PCR analysis showed that the Fyn, Fyn/BAC, and Svet1/IRES-Cre DNA constructs were successfully introduced into Sprague-Dawley rat offspring via ICSI, with production efficiencies of 2.8% (3/109), 0.9% (1/109), and 2.4% (3/125), respectively. These results indicate that transgenic rats can be produced by ICSI-mediated DNA transfer using the various types of exogenous DNA and rat strains with different genetic backgrounds.