A primary culture model of differentiated murine tracheal epithelium.

The goal of this study was to develop a primary culture model of differentiated murine tracheal epithelium. When grown on semipermeable membranes at an air interface, dissociated murine tracheal epithelial cells formed confluent polarized epithelia with high transepithelial resistances ( approximately 12 kOmega. cm(2)) that remained viable for up to 80 days. Immunohistochemistry and light and electron microscopy demonstrated that the cells were epithelial in nature (cytokeratin positive, vimentin and alpha-smooth muscle actin negative) and differentiated to form ciliated and secretory cells from day 8 after seeding onward. With RT-PCR, expression of the cystic fibrosis transmembrane conductance regulator (Cftr) and murine beta-defensin (Defb) genes was detected (Defb-1 was constitutively expressed, whereas Defb-2 expression was induced by exposure to lipopolysaccharide). Finally, Ussing chamber experiments demonstrated an electrophysiological profile compatible with functional amiloride-sensitive sodium channels and cAMP-stimulated CFTR chloride channels. These data indicate that primary cultures of murine tracheal epithelium have many characteristics similar to those of murine tracheal epithelium in vivo. This method will facilitate the establishment of primary cultures of airway epithelium from transgenic mouse models of human diseases.

Enhancing the efficiency of introducing precise mutations into the mouse genome by hit and run gene targeting.

The creation of precise clinical mutations by targeting is important in elucidating disease pathogenesis using mouse models. 'Hit and run' gene targeting is an elegant method to achieve this goal. This uses first a positive selection to introduce the targeting vector carrying the required mutation and then a negative selection to identify clones which have removed vector and wild-type sequences by intrachromosomal recombination. However, this approach has only been successfully used in a handful of cases. We used this procedure to introduce precise clinical mutations into the exon 10 region of the cystic fibrosis transmembrane conductance regulator (Cftr) gene. Using a CMV promoter driven hygromycin/thymidine kinase (hyg/tk) fusion gene as both our dominant and negative selectable marker, we targeted the Cftr locus very efficiently but only identified false runs after the negative selection step. This defect in thymidine kinase induced toxicity to gancyclovir correlated with methylation of the transgene. Consequently we devised a stringent screening procedure to select only true 'run' clones. Unfortunately these 'run' clones had lost the mutation so we altered the vector design to bias the run step to retain the mutation and used a different tk selection cassette with a HSVtk promoter sequence. This new vector design allowed both efficient 'hit and run' for two cystic fibrosis (CF) mutations with no false positives and successful germline transmission of the novel G480C missense mutation.

The mouse bagpipe gene controls development of axial skeleton, skull, and spleen.

The mouse Bapx1 gene is homologous to the Drosophila homeobox containing bagpipe (bap) gene. A shared characteristic of the genes in these two organisms is expression in gut mesoderm. In Drosophila, bap functions to specify the formation of the musculature of the midgut. To determine the function of the mammalian cognate, we targeted a mutation into the Bapx1 locus. Bapx1, similar to Drosophila, does have a conspicuous role in gut mesoderm; however, this appears to be restricted to development of the spleen. In addition, Bapx1 has a major role in the development of the axial skeleton. Loss of Bapx1 affects the distribution of sclerotomal cells, markedly reducing the number that appear ventromedially around the notochord. Subsequently, the structures in the midaxial region, the intervertebral discs, and centra of the vertebral bodies, fail to form. Abnormalities are also found in those bones of the basal skull (basioccipital and basisphenoid bones) associated with the notochord. We postulate that Bapx1 confers the capacity of cells to interact with the notochord, effecting inductive interactions essential for development of the vertebral column and chondrocranium.

Mouse beta defensin-1 is a functional homolog of human beta defensin-1.

Defensin are 3-4 kDa antimicrobial peptides of which three distinct families have been identified; alpha-defensin, beta-defensins, and insect defensins. Recent investigations have shown that beta-defensins are present in the human airways and may be relevant to the pathogenesis of cystic fibrosis (CF) lung disease. We report here the further characterization of a recently identified mouse beta-defensin gene, Defb1, sometimes referred to as mBD-1, which is homologous to the human airway beta defensin hBD-1. We report that Defb1 is expressed in a variety of tissues including the airways and, similar to hBD-1, is not upregulated by lipopolysaccharide (LPS). Defb1 was found to consist of two small exons separated by a 16-kb intron and cytogenetic, and physical mapping linked it to the alpha defensin gene cluster on mouse Chromosome (Chr) 8. Functional studies demonstrate that, like hBD-1, Defb1 demonstrates a salt-sensitive antimicrobial activity against Pseudomonas aeruginosa. Of relevance to CF lung disease is the fact that neither the hBD-1 nor the mBD-1 peptides are active against Burkholderia cepacia.

The mouse Dazla gene encodes a cytoplasmic protein essential for gametogenesis.

RBM and DAZ/SPGY are two families of genes located on the Y chromosome that encode proteins containing RNA-binding motifs, and both have been described as candidate human spermatogenesis genes. Transmission of deletions from father to son has been observed in the case of DAZ, but neither gene family has been shown to be essential for spermatogenesis in human males. The DAZ/SPGY genes are particularly amenable to a knockout approach, as they are found on the Y chromosome in Old World primates and apes, but in other mammals, they are represented only by an autosomal gene, DAZLA, which is also present in Old World primates and apes. It has also been shown that a Dazla homologue is essential for spermatogenesis in Drosophila. Here we show that Dazla protein is cytoplasmic in male and female germ cells, unlike the nuclear RBM protein. Disruption of the Dazla gene leads to loss of germ cells and complete absence of gamete production, demonstrating that Dazla is essential for the differentiation of germ cells.

Short arm dicentric Y chromosome with associated statural defects in a sterile man.

A short arm dicentric Y chromosome is described as the predominant cell line in a sterile man. The patient also presents with short stature. Tooth development appears normal. Only Sertoli cells are present in the seminiferous tubules. It is suggested that the function of the gene controlling spermatogenesis in Yq11 in man might be to prevent proliferation or migration of germ cells to the gonad of the early embryo.

The effect of fluoride on chromosome aberration and sister-chromatid exchange frequencies in cultured human lymphocytes.

Sodium fluoride, at concentrations of up to 60 times the level normally used in drinking water for the prevention of dental decay, was compared with 2 other inorganic salts for its ability to induce chromosome aberrations and sister-chromatid exchanges (SCE) in cultured human lymphocytes. No significant increases in the frequencies of aberrations of SCEs were found.