Biomedical Engineering Reference
In-Depth Information
the scope of this chapter. The foster mother gives birth 17 days later to chimeric pups
derived partly from the injected ES cells and partly from the host embryo.
Chimeras with the potential to transmit the recombinant ES cell genome through the
germline are expected to be male, as they are derived from male R1 ES cells, and can be bred
with C57BL/6 females in order to monitor germline transmission to the resulting offspring
(F1 generation) (Figure 10.5b). Coat color is an indicator of germline transmission; agouti
coat color in F1 mice demonstrates germline transmission, as 129-derived cells yield agouti,
while C57-derived cells yield black pigmentation. PCR and Southern blot screening of
purified tail DNA from agouti F1 animals will determine which F1 animal carries the mutated
allele. Heterozygous F1 animals are then intercrossed to generate homozygous mutant and
wild-type control littermates. In some instances, genetic background can contribute to the
observed phenotype; in this case, animals should be backcrossed to the desired strain (usually
C57BL/6) for more than eight generations so that mutants are maintained and studied on a
pure genetic background.
10.3 Troubleshooting
1
BAC retrieval does not work
•
Use three sets of PCR primer pairs to confirm the presence in the BAC of the two
ends and middle of the sequence to be retrieved.
•
The orientation of the long primers used to amplify the pNAPP vector is critical. Refer
to Figure 10.2 for a schematic.
•
Multiple freeze-thaw cycles compromise the ability of recombineering bacteria to
accept BAC DNA. It may be necessary to obtain a fresh stock of these bacteria.
•
In rare cases, BAC DNA may contain mutations that interfere with BAC retrieval.
These may arise due to low DNA replication fidelity in bacteria. Therefore, we rec-
ommend ordering at least two different BAC clones when performing BAC retrieval.
2
Poor MEF cell yield or morphology
During expansion of MEF cells, make sure the cells are highly confluent before pas-
sage. After 100% confluency is reached, wait 1-2 days before passage.
3
ES cells undergo differentiation
•
•
Ensure that the MEF feeder cells are plated 48 h before ES cells, and that they form
a monolayer over the plate.
•
Ensure that LIF is at the correct concentration and is not past its expiration date.
•
ES cells should be passaged approximately every 3 days; only undifferentiated cells
survive frequent passaging. Refer to Figure 10.4 for images of ES cell confluency and
differentiation.
•
Culture medium should be less than 4 weeks old, as glutamine by-products may be
toxic to ES cells.
•
Slowly growing ES cells are more likely to undergo differentiation. Increase serum
concentration if cells are growing slowly.
