title: Cryopreserved morulae can be used to efficiently generate germline-transmitting chimeras by blastocyst injection.
abstract: The production of chimeric mice is a complex process, requiring the careful coordination of tissue culture cell growth, production of a large number (30-75) of competent blastocysts and the availability of appropriately timed pseudo pregnant female mice. Failure at any of these steps can impinge upon the rapid production of chimeras. One potential improvement for the efficient generation of chimeric mice would be the utilization of cryopreserved embryos suitable for injection. C57Bl/6 morulae were frozen using a standard 2-step protocol with ethylene glycol as the cryopreservation agent. We determined that cryopreserved morulae could thaw, culture to blastocyst stage in KSOM media and survive injection at rates equivalent to control embryos. Cryopreserved morulae were also equivalent to controls at all later stages in the process of production of chimeric mice, including birth rate, percentage chimerism of resulting animals and ability to produce germline progeny. Hence, cryopreservation of morulae for blastocyst injection is a suitable option to enhance the efficiency of chimeric mouse generation.
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Texas M. D. Anderson Cancer Center Genetically Engineered Mouse Facility, 1515 Holcombe Blvd., Houston, Texas, USA. jpthorn@mdanderson.org
authors.forename: Janice V
authors.initials: JV
authors.lastname: Parker-Thornburg
authors.forename: Jennifer L
authors.initials: JL
authors.lastname: Alana
authors.forename: Chad N
authors.initials: CN
authors.lastname: Smith
authors.forename: Michelle
authors.initials: M
authors.lastname: Detry
authors.forename: Marta L
authors.initials: ML
authors.lastname: Rojas
authors.forename: Kedryn K
authors.initials: KK
authors.lastname: Baskin
mesh_terms: D000818:Animals; D001755:Blastocyst; D002678:Chimera / Q000235:genetics*; D015925:Cryopreservation; D004624:Embryo Transfer; D005260:Female; D007267:Injections; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009028:Morula; D011247:Pregnancy