Generating Gene Knockout Mice for Studying Mechanisms Underlying Drug Addiction - Drugs of Abuse: Neurological Reviews and Protocols - page 347

Fig. 1. Strategy for generating a knockout mouse. Genomic DNA locus of a
hypothetical wild-type gene with three exons ( black boxes ); the targeting vector
and the mutant gene locus are indicated. In this particular design, the fi rst exon of
the gene is replaced by a selectable marker, the neo gene, on proper homologous
recombination.

3 arms such that they can be ligated appropriately to generate the targeting

construct, perform gene clean procedure to purify the cloning vector DNA, and
resuspend in TE.

3. Targeting vector construction: Ligate the 5 arms with the cloning vector.

Depending on the ligation strategy, perform either stepwise ligation, which is
usually easier, or four-piece ligation. Transform the ligation mix into the XL1-
Blue bacteria and plate out on LB plates containing ampicillin. Grow individual
colonies at 37°C and isolate the plasmid individually. Digest the plasmid DNA and
perform gel electrophoresis to screen for the targeting construct ( see Note 3 ).

and 3

3.1.3. Quantifi cation and Verifi cation of the Targeting Construct
Once the targeting construct is made, grow 0.5 L of bacteria harboring the
construct. Isolate and purify the plasmid twice by CsCl ultracentrifugation.
Verify the plasmid by restriction enzyme digestion and gel electrophoresis.
Quantify the plasmid by OD 260 reading.

3.1.4. Hybridization Probes
The 5 arms

used in making the targeting construct, detect DNA size changes predicted by
the desired homologous recombination, and detect single-copy DNA sequences
in the genome. Isolate candidate probe DNA fragments and perform genomic
Southern blotting to verify the probes. Alternatively, perform PCR to obtain the
probes and verify by Southern blotting ( see Notes 1 and 4 ).

and 3 probes should ideally be located outside of the 5 and 3