Biology Reference
In-Depth Information
3.5. Mating of Chimeras
To check for germ line transmission, adult chimeric males are mated to 2-3
adult C57B6 females. Transmission of the ES cell genotyope is evidenced by
the birth of agouti pups. The agouti coat color, characteristic of the 129 mouse
strain (from which ES cells derive), is dominant over the black coat color shown
by C57B6 mice. The higher the chimerism, the better the chances for germ line
transmission. If a chimera generates at least three litters of black pups, it is
presumably not able to transmit the ES cell genotype. When chimeras show
about 100% of ES cell contribution, it can happen to obtain agouti pups only.
Transmission of the mutant allele should show a normal mendelian distribu-
tion among the agouti offspring.
Mice obtained by this mating strategy possess a mixed 129 and C57B6
genetic background. An easy way to obtain heterozygous animals of pure 129
inbred strain is to cross the chimera with an adult 129 female. In this case, the
litter obtained shows only the agouti coat color. To identify 129 inbred ani-
mals, it is necessary to test the genotype for the presence of the mutant allele.
Mice that bear the mutation virtually derive from the mating of two 129 inbred
mice and being of pure 129 genetic background, they can be used to generate a
129 inbred mouse colony.
3.6. Genotyping of Offspring
Agouti pups must be genotyped to assess whether they are heterozygous for
the mutation. For this purpose, genomic DNA must be extracted from tail biop-
sies and analyzed for the presence of the targeted allele. The high reliability of
Southern blot techniques makes this it the system of choice for identification
of heterozygotes in the very first litters. Faster but more prone to errors is PCR
analysis, a procedure that is then suitable for genotyping of assessed mutant
mouse strains.
3.6.1. Southern Blot Analysis of Tail DNA
1.
Label 20-30-d-old mice by applying numbered ear clips. Mark number, sex, and
mouse coat color in a notebook for further reference.
2.
Cut the tail with sharp, strong scissors at about 1 cm from the tip and collect the
tissue sample in a labeled sterile 1.5-mL tube. Dissolve tail tissue by incubation
on a rotary wheel with 0.5 mL of tail buffer-PK overnight at 56
°
C.
3.
Add 0.5 mL of phenol-chloroform. Incubate samples for 10 min on the rotary
wheel at RT, then spin them at maximum speed on a bench centrifuge for 5 min.
4.
Collect the DNA containing supernatant, avoiding precipitates at the interface,
with a 1-mL disposable tip (cut to make the opening wider). Add the supernatant
to a fresh tube containing 0.5 mL of chloroform. Incubate samples again on the
rotary wheel for 10 min, then spin them for 5 min at maximum speed.
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