Biology Reference
In-Depth Information
ipulators and micrometer syringes. The whole set-up is mounted on a base-plate
that stands on a vibration-damping table. Additional instruments include a
mechanical puller, a microforge and a diamond-coated grinding wheel; they are
used to prepare the injection and holding pipets. An extensive description of these
instruments and how they should be used can be found in the laboratory manual
by Hogan
et al
.
(9)
(
see
Note 3
).
2.
Recover blastocysts from pregnant C57BL/6J mice 3.5 d
postcoitum
by flushing
the uterine horn with M2 medium. Select only mature blastocysts with large cavi-
ties and keep them in drops of M16 medium under mineral oil at 37
C in the CO
2
incubator. Use the other blastocysts as carriers when reimplanting those injected.
Handling of the animals and surgical procedures are detailed in the aforemen-
tioned manual
(9)
.
°
3.
Seed positive ES clones and expand them in a T
25
flask for 1-2 d and without
selection. Add 0.5 mL of trypsin, incubate at 37
°
C for 4', and neutralize with
10
6
cells/mL. Because cell clumps cannot be
injected, it is imperative to obtain a single-cell suspension. Inject ~12-15 ES
cells/blastocyst in ES medium without selecting agent(s). Store injected blasto-
cysts in M16 medium drops under mineral oil at 37
~2 mL of ES medium at ~1-2
×
°
C in a CO
2
incubator.
4.
Reimplant 6-10 embryos (including two uninjected carrier blastocysts) into one
uterine horn of an anesthetized Swiss-Webster pseudopregnant foster mother.
Cage the animals individually 14 d after the reimplantation to avoid cannibaliza-
tion of the offsprings. At ~2 wk after birth, assess the level of chimerism from the
percentage of agouti color in the newborns' coat (
see
Note 4
).
5.
Cage 5-6-week-old male chimeras with C57Bl6/6J females to test germ-line
transmission of the targeted allele. Extract DNA from 1.5 cm tails of 3-week old
mice. Incubate tails in lysis buffer overnight at 55
C. Remove hair by centrifuga-
tion, add isopropanol, and precipate DNA for ~1 h. After ethanol precipitation
and washing, dissolve DNA in 200
°
µ
L of TE overnight at 55
°
C and then use
10
L of the sample for Southern blot analysis. Once germ-line transmission is
demonstrated, cage the same male chimeras with 129/sv females to bring the
mutation on pure 129/sv genetic background; be aware this cross will yield only
agouti offspring. Alternatively, continue to back-cross the progeny of the [chi-
mera
µ
C57BL/6J] inter-cross for ~10 generations to bring the mutation on
the C57Bl/6J inbred background. While these matings progress, study the
phenotypic and morphologic consequences of the mutation in the randomly mixed
129/sv, C57Bl/6J background.
×
4. Notes
1.
Even if not mentioned in the text, ES cells should always be placed in fresh ES
medium 1 h before being trypsinized and washed with PBS. ES cells should be
picked rather quickly because overtrypsinization reduces germ-line colonization.
To this end, the pipet tips for the picking and processing of ES cells should have
an even opening and fit tightly in the multichannel pipetor. ES colonies are picked
under a standard microscope placed inside the laminar flow hood.
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