Generating Gene Knockout Mice for Studying Mechanisms Underlying Drug Addiction - Drugs of Abuse: Neurological Reviews and Protocols - page 352

3.3.5. Identifi cation of Mice Carrying the Desired Gene
Mutation and Breeding
1. Chimeric mice: At 17-18 d after the blastocyst return, the foster mothers will
give birth to pups. About 10 d after birth, the percent chimerism for individual
pups can be determined. When reaching 6 wk of age, male chimeric mice with a
high percentage of chimerism are bred with C57BL/6J female mice.
2. Germ-line transmission of the desired gene mutation: When the C57BL/6J
females give birth after breeding with the male chimeric mice, the appearance
of the agouti color pups is the fi rst indication that the injected ES cells have
contributed to the germ cell development. To verify this, isolate genomic DNA
from the tail biopsies followed by either Southern blotting or PCR as described
previously.
3. Homozygous knockout mice: Breed heterozygous mice to obtain mice homozy-
gous for the desired deletion. Identify the knockout mice by either Southern
blotting or PCR as described previously ( see Notes 11-15 ).
4. Notes
1. Care should be taken that the right gene has been cloned particularly when
dealing with a gene family. Preferably, DNA sequencing is performed or at least
detailed restriction mapping information is obtained before making the targeting
construct. Moreover, the hybridization probes should be specifi c for the gene of
interest and should be similarly verifi ed.
2. When designing the targeting construct, we prefer to eliminate the translation start
site rather than to insert a sequence in an exon, particularly in the downstream
exons. This helps to eliminate the potential of generating a part of the gene
product or a hybrid gene product in the knockout mice.
3. Other researchers have used a thymidine kinase ( TK ) gene together with the
neo gene in the targeting construct to increase the homologous recombination
rate. To minimize the exposure of ES cells to chemicals, we have not used the
TK gene for selection. In our experience, we get a reasonable recombination
rate with a 5-20% range.
4. If ES cells from the 129 mouse strain will be used to generate the knockout mice,
for maximum homologous recombination rate, genomic library constructed from
DNA isolated from the same mouse strain should be used.
5. For high-quality cell culture work, use sterile and disposable plasticware.
Moreover, use the highest grade chemicals when possible. If making up own cell
culture medium, use double distilled water.
6. Extreme care should be taken to limit potential differentiation of ES cells.
Leukemia inhibitory factor should be added to the ES cell medium and ES cells
should not be overgrown.
7. After the amplifi cation of ES cells, genomic DNA should be analyzed again
by either Southern blotting or PCR to ensure the amplifi ed cells are relatively
homogeneous with respect to the gene mutation.