Expression of laminin chains by central neurons: Analysis with gene and protein trapping techniques

• Published in: Genesis (New York, N.Y. : 2000) N.Y. : 2000), 2003-06, Vol.36 (2), p.114-127
• Main Authors: Yin, Yong, Kikkawa, Yamato, Mudd, Jacqueline L., Skarnes, William C., Sanes, Joshua R., Miner, Jeffrey H.
• Format: Article
• Language: English
• Subjects: Animals; Base Sequence; basement membrane; Blotting, Western; Brain - metabolism; DNA Primers; extracellular matrix; Gene Expression; gene trap; hippocampus; Immunohistochemistry; laminin; Laminin - genetics; Mice; Muscle, Skeletal - cytology; Muscle, Skeletal - metabolism; neomycin phosphotransferase; Neurons - metabolism; Precipitin Tests; retina; transgenes
• ISSN: 1526-954X; 1526-968X
• DOI: 10.1002/gene.10206

Laminins exert numerous effects on neurons in vitro, but expression of laminin subunit genes by neurons in vivo remains controversial. To reexamine this issue, we generated mice from ES cells in which the laminin α1, α5, β1, and γ1 genes had been “trapped” by insertion of a histochemically detectable selectable marker, βgeo (β‐galactosidase fused to neomycin phosphotransferase). The presence of laminin‐βgeo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin β1 and γ1 genes, but not α chain genes, by defined subsets of neurons in brain and retina. We also used the gene traps in a novel way to assay expression of endogenous laminin subunits, which were barely detectable by ordinary immunohistochemical methods. The trapping vector included a transmembrane domain that anchors proteins otherwise destined for secretion. Laminin α/β/γ heterotrimers are assembled intracellularly, and we show that the trapped laminin γ1 fusion protein “co‐trapped” endogenous β1 intracellularly. The laminin γ1 fusion was also able to co‐trap transgene‐derived α chains, but we detected no co‐trapped endogenous α chains. The co‐trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products. genesis 36:114–127, 2003. © 2003 Wiley‐Liss, Inc.