Biomedical Engineering Reference
In-Depth Information
ii
Remember to use ES medium without selection.
jj
Use several dilutions in the final passage to ensure that at least one plate will have an optimal
density of colonies.
kk
Ensure a thorough dissociation of cells in order to allow injection of individual cells.
10.2.5 Mating of chimeras and downstream applications
Homologous recombinant ES cells obtained using the protocols described in this chapter are
microinjected into the fluid-filled blastocoele cavity of 3.5-day-old embryos at the blastocyst
stage; the injected embryos are then surgically implanted in the uterus of pseudopregnant
females (Figure 10.5a). These techniques are described in detail elsewhere and are beyond
(a)
(b)
(d)
X
(c)
Figure 10.5
ES cell injection and chimera breeding. (a) Blastocysts are obtained from a
C57BL/6 mouse (black coat color) and microinjected with targeted R1 ES cells (129 genetic
background: agouti coat color). Injected blastocysts are implanted in a pseudopregnant female
(white mouse). (b) The resulting black and brown male chimeras are then mated with a C57
black female to obtain brown and black F1 offspring (c). Brown pups are then screened by PCR
or Southern blot for germline transmission of the targeted gene. (d) Two adult mouse chimeras
generated through gene targeting in ES cells and injection into a C57BL/6 blastocyst. The mouse
on the left has a higher percentage of agouti coat color; the probability of germline transmission
of the targeted gene to the offspring of this mouse is higher than that for the mouse on
the right. (See Plate 10.5.)
