Figure 1
Figure 1. Generation of mice with an inducible deletion of Cul4A. (A) Restriction maps of the wild-type Cul4A, targeted allele (Cul4A-T), conditional deletion (Cul4A-flox), and Cul4A deletion (Cul4A-Δ1) are shown. Gray boxes denote exons; “ATG” denotes the first coding exon. E indicates EcoRI; K, KpnI; N, NotI; Nh, NheI; P, PstI; and H, HindIII. “H probe” was used for Southern analysis. Dashed lines indicate insertions and deletions. (B) PCR primers used for genotyping. PCR primers (black arrows) flank loxP sites (black triangles) that flank the first coding exon in the conditional Cul4A. (C) Genomic blot to identify Cul4A alleles in mosaic mice. Mutant and wild-type Cul4A alleles are indicated: Δ1NEO indicates deletion of the first coding exon, retention of the floxed Neo (3.7 kb); WT, wild-type Cul4A (3.3 kb); Cul4A-flox, Cul4A conditional deletion (3.3 kb); and Cul4A-Δ1, Cul4A deletion (2.0 kb). Asterisks indicate samples analyzed further by PCR for Cul4A-flox. (D) PCR screen to detect mosaics with Cul4A-flox. Genomic DNA from mosaics identified in the previous screen (C) were screened with PCR primers LOXP2–1 and LOXP2–2. A PCR fragment containing loxP was detected by DNA blot with an oligonucleotide probe. The positive control template (C) was genomic DNA from a Cul4Aflox/+ ES cell line. For panels C and D, representative sample blots are shown. (E) PCR screen to genotype Cul4A-flox mice. PCR primers flanking each loxP site were used to identify wild-type (+/+), Cul4Aflox/+ (flox/+), and Cul4Aflox/flox (flox/flox) mice. (F) Southern analysis to identify Cul4Aflox/+ mice. Genomic DNA from a wild-type mouse (+/+), a Cul4Aflox/+ ES cell line (flox/+ES), and a Cul4Aflox/+ mouse (flox/+mouse) was analyzed by Southern analysis. The doublet in the last 2 lanes consists of the Cul4A wild-type (smaller band) and the Cul4A-flox allele, 116 bp larger with 2 loxP sites. (G) Cul4A deletion is induced in liver and bone marrow. Mutant and control mice were treated with pIpC, and 5 days after induction, DNA from liver, brain, and bone marrow was analyzed by Southern analysis for Cul4A-flox and Cul4A-Δ1. (H) Cul4A protein is not detected in mutant liver after induction of Cre. Ten days after induction, Cul4A was detected by immunoblot in lysates from mutant and control mice. Actin was a loading control.

Generation of mice with an inducible deletion of Cul4A. (A) Restriction maps of the wild-type Cul4A, targeted allele (Cul4A-T), conditional deletion (Cul4A-flox), and Cul4A deletion (Cul4A-Δ1) are shown. Gray boxes denote exons; “ATG” denotes the first coding exon. E indicates EcoRI; K, KpnI; N, NotI; Nh, NheI; P, PstI; and H, HindIII. “H probe” was used for Southern analysis. Dashed lines indicate insertions and deletions. (B) PCR primers used for genotyping. PCR primers (black arrows) flank loxP sites (black triangles) that flank the first coding exon in the conditional Cul4A. (C) Genomic blot to identify Cul4A alleles in mosaic mice. Mutant and wild-type Cul4A alleles are indicated: Δ1NEO indicates deletion of the first coding exon, retention of the floxed Neo (3.7 kb); WT, wild-type Cul4A (3.3 kb); Cul4A-flox, Cul4A conditional deletion (3.3 kb); and Cul4A-Δ1, Cul4A deletion (2.0 kb). Asterisks indicate samples analyzed further by PCR for Cul4A-flox. (D) PCR screen to detect mosaics with Cul4A-flox. Genomic DNA from mosaics identified in the previous screen (C) were screened with PCR primers LOXP2–1 and LOXP2–2. A PCR fragment containing loxP was detected by DNA blot with an oligonucleotide probe. The positive control template (C) was genomic DNA from a Cul4Aflox/+ ES cell line. For panels C and D, representative sample blots are shown. (E) PCR screen to genotype Cul4A-flox mice. PCR primers flanking each loxP site were used to identify wild-type (+/+), Cul4Aflox/+ (flox/+), and Cul4Aflox/flox (flox/flox) mice. (F) Southern analysis to identify Cul4Aflox/+ mice. Genomic DNA from a wild-type mouse (+/+), a Cul4Aflox/+ ES cell line (flox/+ES), and a Cul4Aflox/+ mouse (flox/+mouse) was analyzed by Southern analysis. The doublet in the last 2 lanes consists of the Cul4A wild-type (smaller band) and the Cul4A-flox allele, 116 bp larger with 2 loxP sites. (G) Cul4A deletion is induced in liver and bone marrow. Mutant and control mice were treated with pIpC, and 5 days after induction, DNA from liver, brain, and bone marrow was analyzed by Southern analysis for Cul4A-flox and Cul4A-Δ1. (H) Cul4A protein is not detected in mutant liver after induction of Cre. Ten days after induction, Cul4A was detected by immunoblot in lysates from mutant and control mice. Actin was a loading control.

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