Combining ES Cells with Embryos

The marvel of embryonic stem (ES) cells is that after in vitro culturing and genetic modification, they still have the ability to contribute to the developing embryo, when combined with pre-implantation embryos, to produce chimeras and even completely ES cell-derived animals. In this chapter, we will describe three methods for combining ES cells with embryos: the injection of ES cells into blastocysts, the injection of ES cells into eight-cell stage embryos and aggregation of ES cells with morulae. To date, blastocyst injection is the most commonly used method, adopted by core facilities rather than individual laboratories, partially because of the high cost of equipment and long training period required, prohibitive to some labs. The Injection of eight-cell stage embryos can be performed using the same equipment, but because fewer cells are injected per embryo this method is faster and can be learned quickly by anyone trained in blastocyst injection. The procedure makes use of less expensive outbred embryo donor mice and produces completely ES cell-derived mice when good quality ES cells are used. Morula aggregation is performed under a simple dissecting stereomicroscope, thereby lowering the startup costs, and requires a shorter training period. Although the procedure utilizes less expensive outbred strains of mice as embryo donors, the savings are partially offset by the need for larger numbers of transferred embryos per female due to the lower implantation rate of the zona pellucida (ZP) free embryos. On the other hand, morula aggregations are much faster and easier to perform than microinjections and similar to eight-cell microinjections they often result in fully ES cell-derived animals.

Source: http://www.springerprotocols.com/Abstract/doi/10.1007/978-3-642-20792-1_17