Figure 1.

The Zic2 knockdown mutation. (a) Mouse Zic2 gene, targeting construct, and mutated Zic2 gene (Zic2^kd). The targeting vector (Top) contains an 8.0-kb and a 2.3-kb region homologous to the Zic2 gene and a neomycin-resistance gene, respectively, driven by the phosphoglycerate kinase gene (PGK) promoter (NEO). The diphtheria toxin A fragment gene driven by the MC1 promoter (DT) was inserted in the 3′ end of the Zic2 gene. In the Zic2kd allele, a homologous recombination occurred in the 3′ end, whereas a large portion of the 5′ homologous region (red) was deleted and the remaining part was connected to the first intron (green) illegitimately. The connecting point contains the three bases of the overlapping sequence between the 5′ and intron sequence as determined by nucleotide sequencing (data not shown). As a result, PGKneo and 622 bp of the 5′ homologous regions were inserted into the first intron. (b) Southern blots verifying the structure of the mutated allele. Genomic DNA was extracted from Zic2^+/+, Zic2^kd/+, and Zic2^kd/kd, digested with EcoRI and BamHI, and hybridized with four distinct probes. 5′ and 3′ probes, 5′ and 3′ flanking region of the targeting vector, respectively. Ex1 and NEO probes, fragments containing the Zic2 exon1 and neomycin-resistance gene, respectively. The expected sizes of restriction fragments are indicated in a. (c and d) The amount of Zic2 transcript from E11.5 embryonic tissue was measured by Northern blot (c) or quantitative RT-PCR analysis (d). Expression of the mutated allele was 21% that of the wild-type allele. RNA extracted from E11.5 whole embryo (W), head (H), anterior trunk (AT), and posterior trunk (PT) was analyzed. In c, there are additional bands (*) in Zic2^kd/+ and Zic2^kd/kd, which correspond to the unspliced mRNA precursor (data not shown), in addition to bands that correspond to the mature transcripts (Zic2). Arrowheads indicates the positions of 28S and 18S ribosomal RNA. The bottom frame shows 28S RNA with similar content and integrity. Analysis of RNA from E17.5 embryos gave similar results (data not shown). (e) Immunoblot using the anti-Zic2 antibody (Right). The density of the bands representing Zic2 protein (55.5 kDa) in the E11.5 Zic2^+/+, Zic2^kd/+, and Zic2^kd/kd whole embryos was consistent with the amount of Zic2 transcript shown in c and d. (Left) The total protein used for the immunoblot as revealed by Coomassie blue staining.