An alternative simple method for mass production of chimeric embryos by coculturing denuded embryos and embryonic stem cells in Eppendorf vials.
The generation of germline competent chimeric mice via embryonic stem (ES) cells is a crucial step in developing gene-manipulated mouse models. To date, techniques for generating chimeric mice include direct microinjection of ES cells into the cavity of 3.5-d post-coitum (dpc) blastocysts and aggregating or coculturing 2.5 dpc zona pellucida-free (denuded) embryos with ES cells. We present here a procedure that is simple and reproducible for mass producing (10-150 embryos/vial/time) chimeric embryos by coculturing denuded 8-cell embryos and morula in 0.8 mL KSOM-AA medium containing 5 x 10(5)mL-1 purified green fluorescence protein-expressing ES cells (either fresh or thawed) in an 1.7 mL Eppendorf vial for 3h. The resulting chimeras had substantial levels of chimerism and high germline transmission rates. Therefore, the method developed in this study can provide a simple and mass reproducible alternative method (to germline transmitter chimeric mice), without technological and instrumental difficulties, for generating chimeric embryos.
Lee
Kun-Hsiung
KH
Division of Biotechnology, Animal Technology Institute Taiwan, PO Box 23, Chunan 35099, Miaoli, Taiwan. khlee@mail.atit.org.tw
Chuang
Chin-Kai
CK
Wang
Huei Wun
HW
Stone
Lee
L
Chen
Chien-Hong
CH
Tu
Ching-Fu
CF
D000818:Animals; D030801:Animals, Genetically Modified; D001755:Blastocyst / Q000166:cytology / Q000502:physiology*; D002678:Chimera / Q000235:genetics; D018920:Coculture Techniques / Q000379:methods / Q000662:veterinary; D004624:Embryo Transfer; D053595:Embryonic Stem Cells / Q000166:cytology* / Q000502:physiology; D005260:Female; D005821:Genetic Techniques; D049452:Green Fluorescent Proteins; D008297:Male; D051379:Mice / Q000196:embryology*; D009028:Morula / Q000166:cytology / Q000502:physiology
D049452:Green Fluorescent Proteins