Figure 1
Figure 1. Generation of sclRER/RER knockin mice. (A) Schematic representation of the SCL protein. In black are shown the N-terminal (Nt) and C-terminal (Ct) domains; in gray, the basic (b) helix-loop-helix (HLH) domain. Numbers correspond to amino acid positions. The amino acid sequence shows the 3 residues that are mutated in SCL basic domain (RER, bold and underlined). (B) Strategy for targeted insertion of the mutations into the scl locus. The scl wild-type genomic locus and targeting construct (i), the targeted locus resulting from homologous recombination before (ii, RERneo) and after (iii, RERΔneo) excision of the floxed neo cassette are shown. Solid boxes represent the exons (black indicates noncoding; gray, coding). The domains of SCL (Nt, bHLH, Ct) encoded by exons IV to VI are indicated. Neo, TK indicate neomycin and thymidine kinase selection cassettes, respectively; black triangles, LoxP sites; m: mutation introduced into the sequence coding for SCL basic domain. E, EcoRI; S, SacI. The thin lines above the loci represent the length (in kb) of the restriction fragments detected by Southern blotting. The bold bars under the loci show the position and name (in brackets) of the probes used in the Southern blot analyses. (C) Autoradiograms showing the genomic analysis of wild-type (Wt/Wt) and targeted heterozygous (Wt/RERneo and Wt/RERΔneo) ES clones. The restriction enzymes and probes used are indicated under each blot. (D) Allele-specific PCR analysis. (Left) Schematic representation of the exon coding for SCL bHLH and Ct domains. Homologous recombination introduced a PvuII site in the mutated scl allele. Nucleotide and amino acid sequences are indicated for wild-type and mutated alleles. The PvuII site created by the mutation in the targeted allele is highlighted. (Right) The 418-bp amplified fragment encompassing the mutation gives rise to 2 bands when digested by PvuII. An ethidium bromide–stained agarose gel shows a representative PCR analysis of DNA extracted from wild-type, heterozygous sclWt/RER, and homozygous sclRER/RER cells.

Generation of sclRER/RER knockin mice. (A) Schematic representation of the SCL protein. In black are shown the N-terminal (Nt) and C-terminal (Ct) domains; in gray, the basic (b) helix-loop-helix (HLH) domain. Numbers correspond to amino acid positions. The amino acid sequence shows the 3 residues that are mutated in SCL basic domain (RER, bold and underlined). (B) Strategy for targeted insertion of the mutations into the scl locus. The scl wild-type genomic locus and targeting construct (i), the targeted locus resulting from homologous recombination before (ii, RERneo) and after (iii, RERΔneo) excision of the floxed neo cassette are shown. Solid boxes represent the exons (black indicates noncoding; gray, coding). The domains of SCL (Nt, bHLH, Ct) encoded by exons IV to VI are indicated. Neo, TK indicate neomycin and thymidine kinase selection cassettes, respectively; black triangles, LoxP sites; m: mutation introduced into the sequence coding for SCL basic domain. E, EcoRI; S, SacI. The thin lines above the loci represent the length (in kb) of the restriction fragments detected by Southern blotting. The bold bars under the loci show the position and name (in brackets) of the probes used in the Southern blot analyses. (C) Autoradiograms showing the genomic analysis of wild-type (Wt/Wt) and targeted heterozygous (Wt/RERneo and Wt/RERΔneo) ES clones. The restriction enzymes and probes used are indicated under each blot. (D) Allele-specific PCR analysis. (Left) Schematic representation of the exon coding for SCL bHLH and Ct domains. Homologous recombination introduced a PvuII site in the mutated scl allele. Nucleotide and amino acid sequences are indicated for wild-type and mutated alleles. The PvuII site created by the mutation in the targeted allele is highlighted. (Right) The 418-bp amplified fragment encompassing the mutation gives rise to 2 bands when digested by PvuII. An ethidium bromide–stained agarose gel shows a representative PCR analysis of DNA extracted from wild-type, heterozygous sclWt/RER, and homozygous sclRER/RER cells.

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