Non-intact zona improves development of murine preimplantation embryos transfected by an adenovirus vector

Abstract

The present study explored whether embryos could be transfected by the adenovirus-vector if the zona pellucida (ZP) was not completely removed. An adenovirus vector with green fluorescent protein (pAd-GFP) was used to transfect mouse non-intact zona zygotes (following partial removal of the ZP induced by pronase), zona-free and zona-intact embryos. Non-intact zona and zona-free embryos expressed GFP (confirmed with inverted fluorescence microscopy) after 48 h of culture. The transfection rate of non-intact zona group was up to 51% and the entire zona-free group was transfected. However, none of the zona-intact embryos was transfected. Regardless of whether non-intact zona embryos were transfected by pAd-GFP, their developmental rate (74.3 ± 2.4 and 69.2 ± 3.3% for non-transfected and transfected, respectively; mean ± SEM) was higher (P<0.05) than that of zona-free embryos without and with transfection (54.5 ± 4.3 and 46.7 ± 5.5%). Developmental potential of embryos was decreased for ZP-digestion (non-intact zona 71.8 ± 1.6%; zona-free 50.6 ± 2.2%, P<0.05) or pAd-GFP expression (non-transfected 64.4 ± 1.9%; transfected 56.0 ± 2.1%, P<0.05); therefore, ZP-digestion affected more intensely embryos development than pAd-GFP expression. In summary, non-intact zona murine embryos were readily transfected by the adenovirus-vector, and had much greater development potential than zona-free embryos. Although, the susceptibility of the ZP to digestion by pronase varied among embryos, on average, approximately 3.5 to 4.0 min of digestion resulted in partial removal of the ZP and promoted both transfection and satisfactory embryonic development. It is expected that this method could be used to increase the efficiency of generating transgenic animals.

Keywords: Mouse, non-intact zona embryos, adenovirus vector with green fluorescent protein (pAd-GFP), embryos development