COMPENSATION FOR A GENE TRAP MUTATION IN THE MURINE MICROTUBULE-ASSOCIATED PROTEIN-4 LOCUS BY ALTERNATIVE POLYADENYLATION AND ALTERNATIVE SPLICING

One of the features expected of the gene trap approach is the function al mutation of a gene, allowing its loss-of-function phenotype analysi s. We have mutated the murine microtubule-associated protein 4 (MAP-4) locus by inserting a splice-acceptor gene trap construct. Because the MAP-4 gene has been cloned, sufficient information is available to in vestigate the efficiency of the gene trap insertion in disrupting the protein-coding region. The fusion mRNA contains the first 905 bases of the MAP-4 mRNA and is expected to code for a truncated, nonfunctional MAP-4 protein missing, among others, the microtubule-binding domain. Activity of the lacZ marker gene of the gene trap construct was observ ed in all tissues throughout development and in all cells examined in adult animals. However, some cells and tissues showed higher levels of activity than others: for example, blood vessel endothelium, heart, a spects of the developing nervous system, surface ectoderm of embryonic day 11.5 embryos, and the ependymal layer and blood vessel endotheliu m in adult brain. MAP-4 binds to microtubules and is thought to modula te their stability. It is expressed differentially in different tissue s as 5.5-kb, 6.5-kb, 8-kb, 9-kb, and 10-kb mRNA species from a single copy gene in mice. Northern hybridization with a 5', MAP-4-specific pr obe revealed a 3.3-kb splice variant, which has not been described pre viously, that was expressed as the most abundant MAP-4 mRNA species in the brain but not in other tissues. Mice homozygous for the reported gene trap insertion in the MAP-4 locus (MAP-4(gt/gt)) are viable and a ppear to be phenotypically normal. They exhibited normal levels of all MAP-4 mRNA species in brain and kidney, showing that the simian virus 40-polyadenylation signal of the gene trap construct was ignored and also showing compensation for the gene trap insertion by splicing arou nd the gene trap construct. (C) 1998 Wiley-Liss, Inc.