Insertion Mutagenesis of the Murine RanBP2 Gene
(A) Diagram of the genomic region of RanBP2 disrupted by insertion trap mutagenesis with a bicistronic reporter vector between exon 1 and 2. The bicistronic transcript produces two proteins under regulation of RanBP2. Upon splicing of RanBP2, a fusion between exon 1 and β-geo (a fusion between the β-gal and neo genes) is generated, while human placental alkaline phophatase (PLAP) is independently translated using the internal ribosome entry site. Consistent with previous studies, the expression of the former is directed to cell bodies, while expression of the latter is targeted to the axonal processes [67,68]. Transcriptional 5′ RACE analysis detects a fusion between exon 1 and β-geo.
(B) Southern analysis of the RanBP2 locus of wild-type and heterozygous genomic DNA of tails of F1 mice digested with PpuMI (left panel) and HindIII (right panel) with probes at the 3′ (left panel) and 5′ (right panel) flanking regions of the insertion breakpoint. Q1 is a cosmid containing the RanBP2 gene up to exon 20 [4].
(C) Lateroventral view of a whole-mount stain of a ~12.5 dpc heterozygous embryo for PLAP and β-gal (inset picture) activities. Although PLAP was broadly expressed (e.g., somites, limbs, and CNS), the PLAP and β-Gal (inset picture) expression was particularly high in the optic vesicle (arrow). X-gal single (D) and combined staining with PLAP (E) of a retinal section of a 3-mo-old RanBP2+/− mouse. Consistent with previous immunocytochemistry studies, β-Gal activity is detected in the neuroretinal bodies and inner segment compartment of photoreceptors with conspicuously strong expression in ganglion cells. PLAP expression is found throughout the plexiform/synaptic layers and outer segment of photoreceptors (E). GC, ganglion cell; PLAP, human placental alkaline phophatase; ROS, rod outer segment; RIS, rod inner segment; ONL, outer nuclear layer; OPL, outer plexiform (synaptic) layer; INL, inner nuclear layer; IPL, inner plexiform (synaptic) layer; GC, ganglion cell layer.