Target Discovery and Validation Reviews and Protocols

238 Vijayaraj, Söhl, and Magin
1. Gonadotropin: gonadotropin from pregnant mare’s serum (PMS) (1000 or 5000
U/vial; cat. no. 367222, Calbiochem). Store powder at 4°C. For administration,
the PMS is resuspended at 50 U/mL in sterile 0.9% NaCl and divided into
aliquots.
2. Human chorionic gonadotropin (hCG): chorionic gonadotropin, human urine,
standard grade (1 mg; cat. no. 230734, Calbiochem). hCG is resuspended at final
concentration of 50 U/mL in sterile 0.9% NaCl and divided into aliquots.
3.10.2. Media
KSOM medium complemented with amino acids is recommended to overcome
the inability of two-cell embryos to divide further in culture (two-cell block).
3.11. Superovulating of Females for Preparation of Donor Blastocysts
The consecutive action of two hormones is used to superovulate females.
Gonadotropin from PMS (used to mimic follicle-stimulating hormone) is the
first hormone; hCG (used to mimic luteinizing hormone) is the second hormone.
Strain response to such induction is widely variable. The doses used depend
upon the particular strain and age (or weight) of female and are worked out
empirically. For isolation of blastocysts, we use 4- to 5-wk-old C57BL/6J
females; for collecting oocytes, we use 8- to 10-wk-old FVB females. The times
that the PMS and the hCG are administered are relative to each other and to the
light/dark cycle of the mouse room. For a light/dark cycle of 12 h (6 AM–6 PM),
the optimal isolation protocol is as follows:
1. Injection of the first hormone, PMS, at 12 PM, on day 1:5 U for 4- to 5-wk old
C57BL/6J and 7.5 U for 8- to 10-wk-old FVB.
2. Injection of the second hormone, hCG, 48 h later at 12 PM, on day 3:5 U for 4- to
5-wk-old C57BL/6J and 7.5 U for 8- to 10-wk-old FVB strain mice.
3. Females are mated with normal males after the second injection, one female per
male. A period of 1 or 2 h is normally given for the females to recover from the
stress of injection before mating. Plugs are checked the next morning.
3.11.1. Production of Chimeras by Aggregation
of Diploid With Tetraploid Embryos
The generation of aggregation chimeras has been proven as a powerful tool
for the analysis of genes essential for embryonic development. This technique
can be used to produce a mosaic of two different mice or to restrict the contribution
of one mouse to the extraembryonic or embryonic compartments (132).
Because of the limited developmental potential of tetraploid embryos, their
cells contribute preferentially to the extraembryonic tissues of the embryo,
whereas ES cells are unable to form those compartments. By aggregation of ES
cells and tetraploid embryos, it is possible to generate embryos that consist of
a diploid embryo and a tetraploid extraembryonic tissue. This system has the