Germline mutations in PKD2 cause autosomal dominant polycystic kidney disease. We have introduced a mutant exon 1 in tandem with the wild-type exon 1 at the mouse Pkd2 locus. This is an unstable allele that undergoes somatic inactivation by intragenic homologous recombination to produce a true null allele. Mice heterozygous and homozygous for this mutation, as well as Pkd+/- mice, develop polycystic kidney and liver lesions that are indistinguishable from the human phenotype. In all cases, renal cysts arise from renal tubular cells that lose the capacity to produce Pkd2 protein. Somatic loss of Pkd2 expression is both necessary and sufficient for renal cyst formation in ADPKD, suggesting that PKD2 occurs by a cellular recessive mechanism.

We describe a novel cloning method termed SLiCE (Seamless Ligation Cloning Extract) that utilizes easy to generate bacterial cell extracts to assemble multiple DNA fragments into recombinant DNA molecules in a single in vitro recombination reaction. SLiCE overcomes the sequence limitations of traditional cloning methods, facilitates seamless cloning by recombining short end homologies (≥15 bp) with or without flanking heterologous sequences and provides an effective strategy for directional subcloning of DNA fragments from Bacteria Artificial Chromosomes (BACs) or other sources. SLiCE is highly cost effective as a number of standard laboratory bacterial strains can serve as sources for SLiCE extract. In addition, the cloning efficiencies and capabilities of these strains can be greatly improved by simple genetic modifications. As an example, we modified the DH10B Escherichia coli strain to express an optimized λ prophage Red recombination system. This strain, termed PPY, facilitates SLiCE with very high efficiencies and demonstrates the versatility of the method.

Ovarian failure leading to infertility can be caused by improper prenatal development of the fetal gonad or disruption of the complex postnatal process of folliculogenesis due to alterations in intragonadal or extragonadal regulation. It is critical to have physiological models that mimic events occurring during human development to understand, treat, and prevent ovarian failure in women. Many workers have chosen the mouse as the mammalian model with which to study ovarian function. This review summarizes several key events in female gonadogenesis and folliculogenesis in mice with specific emphasis on spontaneous or induced mutations yielding mouse models that have female infertility owing to ovarian failure. 184 J. A. Elvin and M. M. Matzuk Cohen P, Zhu L and Pollard J (1997) Absence of colony stimulating factor-1 in osteopetrotic (csfm op /csfm op) mice disrupts estrous cycles and ovulation Biology of Reproduction 56 110-118 *Colledge W, Carlton M, Udy G and Evans M (1994) Disruption of c-mos causes parthenogenetic development of unfertilized mouse eggs Nature 370 65-68 Coulombre J and Russell E (1954) Analysis of the pleiotropism at the W-locus in the mouse: the effects of W and W v substitution upon postnatal development of germ cells Journal of Experimental Zoology 126 277-296 Couse J, Curtis S, Washburn T, Lindzey J, Golding T, Lubahn D, Smithies O and Korach K (1995) Analysis of transcription and oestrogen insensitivity in the female mouse after targeted disruption of the oestro-gen receptor gene Molecular Endocrinology 9 1441-1454 Dinchuk J, Car B, Focht R, Johnston J, Jaffee B, Covington M, Contel N, Eng V, Collins R, Czerniak P, Gorry S and Trzaskos J (1995) Renal abnormalities and an altered inflammatory response in mice lacking cyclooxygenase II Nature 378 406-409 *Dong J, Albertini D, Nishimori K, Kumar T, Lu N and Matzuk M (1996) Growth differentiation factor-9 is required during early ovarian folliculo-genesis Nature 383 531-535

ABSTRACT

Caveolin-3, a muscle-specific caveolin-related protein, is the principal structural protein of caveolae membrane domains in striated muscle cells. Recently, we identified a novel autosomal dominant form of limbgirdle muscular dystrophy (LGMD-1C) in humans that is due to mutations within the coding sequence of the human caveolin-3 gene (3p25). These LGMD-1C mutations lead to an ϳ95% reduction in caveolin-3 protein expression, i.e. a caveolin-3 deficiency. Here, we created a caveolin-3 null (CAV3 ؊/؊) mouse model, using standard homologous recombination techniques, to mimic a caveolin-3 deficiency. We show that these mice lack caveolin-3 protein expression and sarcolemmal caveolae membranes. In addition, analysis of skeletal muscle tissue from these caveolin-3 null mice reveals: (i) mild myopathic changes; (ii) an exclusion of the dystrophin-glycoprotein complex from lipid raft domains; and (iii) abnormalities in the organization of the T-tubule system, with dilated and longitudinally oriented T-tubules. These results have clear mechanistic implications for understanding the pathogenesis of LGMD-1C at a molecular level.