Figure 1. Creation of conditional and disrupted NuMA alleles. (A) Schematic representations of (i) a portion of the mouse NuMA gene including exons 16–25 and EcoRI restriction sites, (ii) the exon 22 targeting vector showing the neomycin resistance (Neo) and diphtheria toxin (DT) cassettes and placement of loxP and FRT sites, (iii) the structure of the correctly targeted allele with the introduced SacII restriction site and locations of genotyping primers, (iv) the conditional allele (flox) produced by Flp-enhanced recombinase–mediated recombination of FRT sites flanking Neo, and (v) the deletion allele (Δ) produced by Cre recombination of loxP sites surrounding exon 22. Red bars indicate exon 22. (B) Genomic DNA blotting from neomycin-resistant ES clones after digestion with either EcoRI alone or EcoRI and SacII in combination and hybridization with the 5′ probe shown in A. (C) Predicted PCR fragment sizes for wild-type, Neo, flox, and Δ22 alleles of NuMA using primer sets shown in A. (D) PCR products from neomycin-resistant ES clones using primers i, ii, and iv. (E) PCR products from mouse tail DNA using primers i, ii, and iii. (F) Four independently targeted ES clones and a dilution series blotted with antibodies to NuMA and tubulin. NR, nonrecombined; HR, homologously recombined.
Figure 1.

Creation of conditional and disrupted NuMA alleles. (A) Schematic representations of (i) a portion of the mouse NuMA gene including exons 16–25 and EcoRI restriction sites, (ii) the exon 22 targeting vector showing the neomycin resistance (Neo) and diphtheria toxin (DT) cassettes and placement of loxP and FRT sites, (iii) the structure of the correctly targeted allele with the introduced SacII restriction site and locations of genotyping primers, (iv) the conditional allele (flox) produced by Flp-enhanced recombinase–mediated recombination of FRT sites flanking Neo, and (v) the deletion allele (Δ) produced by Cre recombination of loxP sites surrounding exon 22. Red bars indicate exon 22. (B) Genomic DNA blotting from neomycin-resistant ES clones after digestion with either EcoRI alone or EcoRI and SacII in combination and hybridization with the 5′ probe shown in A. (C) Predicted PCR fragment sizes for wild-type, Neo, flox, and Δ22 alleles of NuMA using primer sets shown in A. (D) PCR products from neomycin-resistant ES clones using primers i, ii, and iv. (E) PCR products from mouse tail DNA using primers i, ii, and iii. (F) Four independently targeted ES clones and a dilution series blotted with antibodies to NuMA and tubulin. NR, nonrecombined; HR, homologously recombined.

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