The availability of gene constructs for various extracellular matrix molecules, makes it now possible to test the function of these molecules, the regulation of their expression, and their use in gene replacement therapy by incorporating these constructs into mouse eggs, thus creating transgenic mice. A transgenic mouse facility has been established and staff have been trained to carry out timed breeding of donor and foster mothers. Several different strains have been tested for oocyte suitability for injections. The complex techniques involved in DNA injection have been mastered and injections are carried out on a daily routine. The capacity of the facility will be doubled in 1987. We are starting to use the collagen II constructs in which various lengths of the 5' flanking region are coupled to the gene for chloramphenicol acetyl transferase (CAT) with or without putative enhancer sequences. The DNA construct is injected into the male pronucleus of fertilized oocytes which are transferred to pseudo-pregnant foster females. Viable young which have incorporated the injected DNA into their chromosomes are then assayed for CAT activity to examine tissue specific expression. The BS-CAT construct (2 Kb of the 5' flanking DNA from rat collagen II gene fused to CAT) has been successfully established as a stable transgenic line but has not shown the expected tissue expression. We anticipate that this system will be useful in testing recombinant constructs of cartilage and basement membrane genes as possible models for gene therapy. When full length cartilage proteoglycans are available, we plan to use mutant (CMD) mice unable to produce cartilage specific proteoglycan as a test system. Other mutant mouse lines such as congenital and juvenile polycystic kidney disease, hypochondroplasia, chondrodysplasia, progressive ankylosis, and fragilitas ossium are potential targets for this type of genetic intervention when the molecular basis of their defects is determined.
