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. Author manuscript; available in PMC: 2012 Dec 1.
Published in final edited form as: J Neuropathol Exp Neurol. 2011 Dec;70(12):1124–1137. doi: 10.1097/NEN.0b013e31823b0b12

Figure 3.

Figure 3

Deletion of short isoform (SN)-Agrn. (A) The single exon encoding the SN amino terminus of AGRIN and approximately 2 kb of upstream sequence were targeted by homologous recombination; upstream exons encoding the long isoform (LN)-N-terminus and downstream exons encoding common sequences were left intact. The loxP-flanked selectable marker, Neo, for embryonic stem cells was removed from the genome by mating to Cre transgenic mice and a single loxP site and flanking restriction sites were left in the Agrn gene. Southern blotting DNA samples from control and homozygous SN-deletion mice confirmed the homologous recombination event with the anticipated shifts in restriction fragment sizes. (B) In situ hybridization with a probe specific to the SN-isoform of Agrn revealed strong expression in the adult hippocampus. (C) Hybridization with a probe recognizing all Agrn isoforms showed a similar pattern of expression. D, E: Hybridization with SN-specific (D) or pan-Agrn (E) probes indicated a lack of Agrn expression in the hippocampus of adult SN-Agrn knockout mice, indicating that SN-Agrn is the predominant isoform expressed in the hippocampus. (F, G) Adult neuromuscular junctions stained with α-bungarotoxin to label AChRs (red) and an antibody against the C-terminus of AGRIN (green) revealed no changed in AGRIN localization or NMJ morphology in the SN-Agrn knockout mice. (H, I) Kidney glomeruli stained with an antibody against the C-terminus of AGRIN revealed abundant basement membrane staining in both control (H) and SN-Agrn knockout (I) kidneys. Data in panels F–I are consistent with preserved expression of LN- AGRIN. (J) SN-AGRIN is the predominant isoform in brain. In AChR clustering activity assays, homogenates from SN-Agrn knockout brains were reduced in clustering activity by 4- to 5-fold compared to homogenates from control brains (n = 3 mice of each genotype), indicating SN-AGRIN is the predominant Z+ isoform of AGRIN expressed by CNS neurons. Background AChR clustering activity was determined by treating myotube cultures with recombinant inactive AGRIN and 100% activity was determined using saturating doses of recombinant Z8 AGRIN.