Creation of gene targeted mutations and the introduction of dominant mutations in transgenic mice are useful for understanding the function of proteins in development and for screening animal models of various diseases for potential therapies. The purpose of this project is to create animal models in mice for studying the molecular basis of genetic and acquire diseases associated with connective tissues. These models will also be useful in elucidating the role of these proteins in development. Genes for the basement membrane and cartilage components have been cloned and the exon-intron structure of these gene have been characterized. Mouse laminin alpha2, beta3, and gamma2 chains have been cloned and sequenced. Murine dystrophia muscularis-2J (dy2J) is an autosomal recessive disease characterized by muscle degeneration and developmental dysmyelination of peripheral nerves. We have identified the defect in the laminin alpha2 chain of dy2J mice. The alpha2-chain cDNA sequence, amplified by RT-PCR from dy2J mice identified a novel, predominant transcript with a 171 base in-frame deletion. This was confirmed with a splice donor site mutation in the alpha2 chain gene of dy2J mice. Translation of this variant transcript would result in the expression of a truncated alpha2 chain with a 57 amino acid deletion and also a substitution of Gln by Glu at residue 91 in the N-terminal domain VI, which is presumed to be involved in the self aggregation of laminin and in cell attachment. The mutant alpha2 chain could disrupt the formation of the laminin network or the binding to Schwann cells and thereby lead to muscle degeneration. To examine cell-type specific activity of the type II collagen enhancer and promoter in vivo, We have prepared lacZ reporter gene constructs with combinations of various sizes of the type II promoter and enhancer containing intron fragments and have micro injected them into mouse embryos to create transgenic mice. We have established homozygous mouse lines with most of these constructs and have been analyzing expression patterns of the lacZ gene in these mice. Mutations in the endogenous genes for basement membrane and cartilage proteins has been attempted by homologous recombination.
