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. 1999 Nov 1;19(21):9298–9305. doi: 10.1523/JNEUROSCI.19-21-09298.1999

Fig. 1.

Fig. 1.

Gene targeting of the neuronal nAChR β2 and β4 subunits. A, Partial genomic structure of the murine β2 gene including the region from exons 1 to 6 (solidboxes) and the structure of the targeting vector are shown. The targeting vector contains Neo as a positive selectable marker. A homologous recombination event generated the deletion from exons 1 to 5. The diagnostic probe at the 5′-flanking region is shown as an openbox.B, Partial genomic structure of the murine β4 gene and β4-targeting vector are depicted with restriction enzyme sites and exons 5 and 6 (solidboxes). The targeting vector contains a puromycin resistance cassette (Puro) as a positive selectable marker and the Herpes simplex thymidine kinase gene (TK) as a negative selectable marker to obtain a replacement mutation. The diagnostic flanking probe is indicated as an openbox. In both A and B the restriction enzyme sites are as follows: E,EcoRI; H,HindIII;P,PstI; S,SacI; Sa,SalI; andX, XhoI. C, The three breeding schemes used to generate the mice studied are shown. Left, β2+/+β4−/− were compared with β2−/−β4−/− littermates.Center, Similarly, this breeding allowed comparison of β2−/−β4+/+ mice with β2−/−β4−/− littermates.Right, Finally, in this breeding, β2+/+β4−/− mice were compared with β2+/+β4+/+ (wild-type) littermates.D, Southern blot analysis identified the mice with the indicated genotypes using β2- and β4-flanking probes as indicated in A and B. E, Northern blot analysis of the expression of β2 mRNA in the brains of β2+/+β4+/+, β2−/−β4+/+, β2+/+β4−/−, and β2−/−β4−/− mice is shown. The probes are the rat cDNA of β2 and a cDNA for the control gene glyceraldehyde-3-phosphate dehydrogenase (gapdh). F, Reverse transcription-PCR analysis of β4 expression in the brains of β2+/+β4+/+, β2−/−β4+/+, β2+/+β4−/−, and β2−/−β4−/− mice is shown. Reverse transcription was performed with the addition of reverse transcriptase (RT) or absence of the enzyme (−) as the negative control, followed by PCR with primers of either the β4 gene or the hprt gene. The size of the PCR product is 176 bp for the β4 gene and 266 bp for the hprt gene.