International Mammalian Genome Society

The 14th International Mouse Genome Conference (2000)


D10. Optimisation of Approaches to the Generation of Chimaeric Mice Using Embryonic Stem Cells.

Elizabeth Williams, Judy Hallett & David Hume
Transgenic Animal Service Queensland, Institute for Molecular Bioscience, University of Queensland, Q4072, Australia (www.cmcb.uq.edu.au/TASQ)

The use of homologous recombination as an approach to targeted gene modification in mouse embryonic stem cells (ES cells) has rapidly advanced the study of gene function using mice as model mammals. With the ongoing success of this technology comes an ever-increasing demand on transgenic core facilities to produce chimaeric mice efficiently and cost-effectively. Although chimaeras can be obtained from some ES cell lines by direct aggregation with stripped morulae, the most common method is by injection of targeted ES cells into blastocysts. The "science" of micro-injection is constrained by the timing of mouse mating, fertilization and gestation. The optimal stage for ES cell injection is believed to be an expanded blastocyst. The majority of core facilities offering the service of blastocyst injection will guarantee a certain number of blastocysts injected/session. As the timing of superovulation and mating is not entirely reliable in any animal facility, the transgenic facility can be forced to cancel sessions when the yield of injectable blastocysts is low, due to either a delayed or too advanced developmental stage. To obviate the problem with delayed embryos, many facilities will culture them overnight and inject the blastocysts the following day. The final preparation of the ES cells on the day of injection can also be time consuming, potentially leading to the wastage of blastocysts which have become too advanced to inject.

In this study, we sought to expand the window of time for the injection of ES cells into mouse embryos. We have successfully produced chimaeric mice by the injection of targeted ES cells into the compacted morulae prior to the formation of the blastocoel cavity, thereby increasing the window of time by 12hrs. To expedite ES cell preparation, we have developed a revised method which involves selecting colonies directly from the culture plate and disaggregating them in a microfuge tube. The combination of these two methods results in a reduction in the time and workload necessary for an injection technician in the preparation and injection of ES cells. Benefits to the transgenic core facility include the reduction in the numbers of mice and sessions (and cost to researchers) needed to create successful chimaeras.

 


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