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

Abstract

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 alpha1, alpha5, beta1, and gamma1 genes had been "trapped" by insertion of a histochemically detectable selectable marker, betageo (beta-galactosidase fused to neomycin phosphotransferase). The presence of laminin-betageo fusion proteins was assayed histochemically and immunochemically, revealing expression of laminin beta1 and gamma1 genes, but not alpha 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 alpha/beta/gamma heterotrimers are assembled intracellularly, and we show that the trapped laminin gamma1 fusion protein "co-trapped" endogenous beta1 intracellularly. The laminin gamma1 fusion was also able to co-trap transgene-derived alpha chains, but we detected no co-trapped endogenous alpha chains. The co-trapping method may be generally useful for identifying proteins or isolating protein complexes associated with trapped gene products.

Disruption of Glomerular Basement Membrane Charge through Podocyte-Specific Mutation of Agrin Does Not Alter Glomerular Permselectivity

“…This is expected to be membrane-bound in the ER in a configuration that exposes the NtA to the lumen where it would be free to interact with its only known binding partner, laminin ␥1; we hypothesize that this leads to "co-trapping" of laminin trimers containing ␥1, as previously shown. 45 In contrast, nidogen-1 and the type IV collagen chains of mature GBM (␣3, ␣4, and ␣5) were detected in the normal pattern and at normal levels in agrin-deficient mice ( Figure 5, C and D; results not shown). All of these BM components In podocyte-specific knockouts up to ϳ7 months of age (D), staining for perlecan is properly localized to the mesangium and indistinguishable from controls (C).…”

Compositional and structural requirements for laminin and basement membranes during mouse embryo implantation and gastrulation

“…We obtained the Lama1 and Lamb1 gene trap ES cell lines and produced mice carrying the insertions (Yin et al, 2003). Based on 5′ RACE analysis and comparisons with the Celera Genomics Mouse Genome Assembly, we determined that in the Lama1 trap line the gene trap vector (pGT0TMp) inserted in the 20.4 kb third intron of Lama1 to produce a fusion protein containing the first 120 amino acids of laminin α1 (part of domain VI), the vector-encoded TM domain and βgeo ( Fig.…”

“…Based on 5′ RACE analysis and comparisons with the Celera Genomics Mouse Genome Assembly, we determined that in the Lama1 trap line the gene trap vector (pGT0TMp) inserted in the 20.4 kb third intron of Lama1 to produce a fusion protein containing the first 120 amino acids of laminin α1 (part of domain VI), the vector-encoded TM domain and βgeo ( Fig. 1A) (Yin et al, 2003). In the Lamb1 trap line, the vector (pGT1TMpfs) inserted into the 10.2 kb 23rd intron to produce a fusion protein containing the first 1178 amino acids of laminin β1 (domains III through VI), the TM domain and βgeo ( Fig.…”

“…Their isolation and identification and production of heterozygous mice have been reported Mitchell et al, 2001;Yin et al, 2003). Generation of Lama5 -/-mice has been previously described (Miner et al, 1998).…”

Schwann Cell-Specific Ablation of Laminin γ1 Causes Apoptosis and Prevents Proliferation

“…Five ␣-chains, four ␤-chains, and three ␥-chains have been identified, and 15 isoforms have been observed (Yin et al, 2003). Laminin ␥1 is one of the most abundant chains and is present in 11 isoforms, including all known laminin isoforms expressed in the PNS (Patton et al, 1997;Previtali et al, 2003).…”