Generating Gene Knockout Mice for Studying Mechanisms Underlying Drug Addiction - Drugs of Abuse: Neurological Reviews and Protocols - page 353

8. Before the blastocyst injection, a mycoplasma test is recommended to ensure that
the ES cells are not contaminated, which will prevent germ-line transmission.
9. During the blastocyst injection, choose the best ES cells that are not differentiated
and are with a round morphology and sharp edges. Do not choose those that have
a fl at morphology and fussy edges.
10. It is diffi cult to predict which ES cell clone will give rise to mice with a high
percentage of chimerism and germ-line transmission. To increase the chance of
germ-line transmission, multiple ES clones should be injected.
11. Maintain a pathogen-free mouse colony.
12. Mice with different genetic background can perform differently, particularly in
behavioral studies. Depending on the goal of the analysis, littermate controls
should be used. Preferably and eventually, one will need to breed the knockout
mice back into a pure genetic background of choice.
13. Because developmental compensations can occur during mouse development,
care should be taken to check for any detectable defi ciencies in the knockout
mice. Moreover, caution should be exercised in data interpretation. Conditional
gene targeting may help to overcome such compensations.
14. Timetable: Targeting construct: 1-3 mo. Homologous recombinants: 2 mo.
Knockout mice: 6-7 mo.
15. After obtaining the knockout mice, one will need to compare the expression of
gene of interest in the knockout mouse and normal control mouse. Moreover,
we recommend performing a general development and behavioral survey of
the knockout mouse. Then, depending on the scientifi c questions asked, one
can combine behavioral, electrophysiological, neuroanatomical, and molecular
biological approaches to analyze the knockout mice. The combined use of
genetically engineered mouse models with proper analyses has tremendous
potential to provide novel insights into mechanisms underlying drug addiction.
Acknowledgment
J. Z. and M. X. were supported by grants from the NIH, the U.S. Army, and
the Epilepsy Foundation of America.
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