Intraoviductal introduction of plasmid DNA and subsequent electroporation for efficient in vivo gene transfer to murine oviductal epithelium.
Various growth factors and proteins produced by oviductal cells have been demonstrated to interact with developing embryos. However, little is known concerning the function of mammalian oviducts at the molecular biological level. This may be partly due to lack of efficient gene transfer to oviductal cells. In this study, we developed an efficient method for transfection of oviductal epithelium using in vivo electroporation (EP) in mice. One microliter of solution containing enhanced green fluorescent protein (EGFP) expression plasmid (0.5 microg) and 0.05% trypan blue (TB) were directly introduced into the ampulla of the eCG-hCG-treated B6C3F1 females at embryonic day (E) 0.6 of pregnancy (corresponding to 14:00-15:00 of the day the plug was recognized). The entire oviduct was then electroporated using tweezer-type electrodes attached to a T820 electroporator (BTX Genetronics, Inc., San Diego, CA) with eight square-wave pulses, 50 V in strength and 50 msec in duration. On E 3.4, embryos at morula/early blastocyst stages were collected and their number, morphology, and EGFP-derived fluorescence recorded. Fluorescence in oviducts was also examined. In some cases, these fluorescent oviducts were subjected to cryostat sectioning. Strong fluorescence was observed in some of the oviductal epithelia, with a maximum level of 36%. Neither the number nor morphology of the collected embryos was affected by EP. Some embryos possessed fluorescence in the blastocoel, but not cytoplasm, suggesting incorporation of EGFP present in the oviductal luminal fluid. This system may enable development of new factors regulating development of preimplantation embryos and offers the prospect of a new approach to understanding oviductal function.
Sato
Masahiro
M
The Institute of Medical Sciences, Tokai University, Bohseidai, Isehara, Kanagawa, Japan. masasato@is.icc.u-tokai.ac.jp
D000818:Animals; D018274:Electroporation / Q000379:methods*; D004848:Epithelium / Q000502:physiology; D005187:Fallopian Tubes / Q000166:cytology / Q000502:physiology; D005260:Female; D018014:Gene Transfer Techniques; D051379:Mice; D010957:Plasmids / Q000008:administration & dosage*