Use of cryopreserved pronuclear embryos for the production of transgenic mice

A series of experiments was conducted to test the hypothesis that an improv ed cryopreservation protocol for pronuclear stage mouse embryos will produc e transgenic (Tg) mice by pronuclear gene injection at a rate not significa ntly different from noncryopreserved embryos. In the first experiment, thre e cryoprotective agents (CPAs) (dimethyl sulfoxide [DMSO], propylene glycol [PG], ethylene glycol [EG]) and two cryopreservation protocols, currently used for pronuclear embryos, were compared in regard to their ability to ma intain post-thaw morphological integrity and in vitro developmental compete nce. In the second and third experiments, the optimal cryopreservation prot ocol determined from the first experiment was used to evaluate in vitro dev elopmental competence of pronuclear embryos following green fluorescence pr otein gene injection and in vivo developmental competence as well as the ge ne integration rates. Survival (morphological integrity and development to two cells) of embryos cryopreserved in the presence of DMSO was higher (P < 0.05) than those cryopreserved with either PG or EG. Postinjection develop mental competence (development to two cells) of cryopreserved CBA, C57B6/Jx CBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreser ved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserve d CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuse s between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C 57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronucl ear embryos can be a useful and efficient approach to the production of Tg mice.