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. 2008 Feb 6;3(2):e1533. doi: 10.1371/journal.pone.0001533

Figure 2. Generation of knock-in mice for the SPIG1 gene and expression of GFP in the retina.

Figure 2

A, Schematic representation of the structure of the endogenous allele (genomic), targeting vector (vector), and targeted allele (targeted). The genomic sequence of the head of exon 2 and the corresponding amino acid sequence in the targeted allele are shown below. The first coding exon of SPIG1 is indicated by blue boxes. For construction of the targeting vector, a gfp-Neo cassette was inserted in frame in the signal sequence of SPIG1 after the first four amino acids, Met-Lys-Pro-Gly, to yield fusion to the N-terminus of GFP through the linker sequence. The DT-A cassette was placed at the 3′ terminus of the homologous region for negative selection. The region used as a probe for Southern blotting is indicated by a bold bar. The position of primer sequences used for PCR analysis is also shown by arrowheads. B, Southern blot analysis of ApaI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. C, Genomic PCR analysis of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. D, RT-PCR analysis of total RNA from the brain of wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. E, Southern blot analysis of PstI- or KpnI-digested genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice with a probe for the coding region of gfp. F, Expression of GFP in the retina of SPIG1gfp/+ mice in a cross section at P5. Enlargements of the boxed regions are shown on the right. In the ventral area, GFP is expressed only in a small subset of cells in the GCL (arrows). In a cross section, dorsal (D) is upwards, and ventral (V) is downwards. inl, inner nuclear layer; onl, outer nuclear layer. G, Expression of GFP in the retina of SPIG1gfp/+ mice in a flat-mount at P5. The retina is apparently subdivided into two domains with SPIG1 expression, the dorsotemporal and pan-ventronasal domains. Enlargements of the boxed regions are shown on the right. d, dorsal; n, nasal; t, temporal; v, ventral. In the pan-ventronasal domain, SPIG1-expressing cells form a regular mosaic. H, Double staining of GFP and BrdU in a cross section at E15.5. Enlargements of the boxed regions are shown on the right. GFP is not expressed in the proliferating cells at S phase. rpe, retinal pigment epithelium. Scale bars: 200 µm (left panels in F/H); 50 µm (enlargements in F/G, and H); 1 mm (left panel in G).