Characterization of the ICSI‐mediated gene transfer method in the production of transgenic pigs

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Understanding the behavior of transgenes introduced into oocytes or embryos is essential for evaluating the methodologies for transgenic animal production. We investigated the expression pattern of a transgene transferred to porcine eggs by intracytoplasmic sperm injection-mediated gene transfer (ICSI-MGT) or pronuclear microinjection (PN injection). The introduction of the EGFP gene by ICSI-MGT yielded significantly more embryos with non-mosaic transgene expression (P < 0.01). In the ICSI-MGT group, 61.5% (24/39) of the embryos were EGFP-positive in all their component blastomeres at the morula stage, while fewer than 10% of such embryos were EGFP-positive in the PN-injection group. Using three types of transgenes, ranging from 3.0 to 7.5 kb in size, we confirmed that approximately one in four fetuses obtained by ICSI-MGT was transgenic, suggesting that ICSI-MGT is a practical method for transgenic pig production. Southern blot analysis of 12 transgenic fetuses produced by ICSI-MGT revealed that the number of integrated transgene copies varied from 1 to 300, with no correlation between transgene size and the number of integrated copies. Fluorescence in situ hybridization analysis revealed that the transgenes were randomly integrated into a single site on the host chromosomes. Together, these data indicate that multiple-copy, single-site integration of a transgene is the primary outcome of ICSI-MGT in the pig and that ICSI-MGT is less likely than PN injection to cause transgene integration in a mosaic manner.