title: Use of cryopreserved pronuclear embryos for the production of transgenic mice.
abstract: A series of experiments was conducted to test the hypothesis that an improved cryopreservation protocol for pronuclear stage mouse embryos will produce transgenic (Tg) mice by pronuclear gene injection at a rate not significantly different from noncryopreserved embryos. In the first experiment, three 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 maintain post-thaw morphological integrity and in vitro developmental competence. In the second and third experiments, the optimal cryopreservation protocol determined from the first experiment was used to evaluate in vitro developmental competence of pronuclear embryos following green fluorescence protein gene injection and in vivo developmental competence as well as the gene 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 developmental competence (development to two cells) of cryopreserved CBA, C57B6/JxCBA-F1 and noncryopreserved (control) embryos was not different (P > 0.05). Postinjection blastocyst formation rate of cryopreserved and noncryopreserved C57B6/JxCBA-F1 embryos was similar (P > 0.05); however, noncryopreserved CBA embryos resulted in a higher blastocyst formation than controls (P < 0.05). While there was no difference in the percentage of transgenic fetuses between cryopreserved and control CBA embryos (P > 0.05), cryopreserved C57B6/JxCBA-F1 embryos resulted in lower transgenic fetuses than control (P < 0.05). These results indicate that the use of cryopreserved mouse pronuclear embryos can be a useful and efficient approach to the production of Tg mice.
authors.affiliation: Institute of Molecular Medicine and Genetics, Medical College of Georgia, Augusta, Georgia 30912, USA.
authors.forename: L
authors.initials: L
authors.lastname: Keskintepe
authors.forename: Y
authors.initials: Y
authors.lastname: Agca
authors.forename: G A
authors.initials: GA
authors.lastname: Pacholczyk
authors.forename: A
authors.initials: A
authors.lastname: Machnicka
authors.forename: J K
authors.initials: JK
authors.lastname: Critser
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000502:physiology; D002467:Cell Nucleus; D015925:Cryopreservation; D003451:Cryoprotective Agents; D004121:Dimethyl Sulfoxide; D004622:Embryo, Mammalian / Q000502:physiology / Q000648:ultrastructure; D019855:Ethylene Glycol; D018014:Gene Transfer Techniques; D051379:Mice; D008810:Mice, Inbred C57BL; D008808:Mice, Inbred CBA; D008845:Microinjections; D019946:Propylene Glycol
chemical_list: D003451:Cryoprotective Agents; D019946:Propylene Glycol; D019855:Ethylene Glycol; D004121:Dimethyl Sulfoxide