Inactivation of the FGF-4 gene in embryonic stem cells alters the growth and/or the survival of their early differentiated progeny.

Previous studies have shown that early mouse embryos with both FGF-4 alleles inactivated are developmentally arrested shortly after implantation. To understand the roles of FGF-4 during early development, we prepared genetically engineered embryonic stem (ES) cells, which are unable to produce FGF-4. Specifically, we describe the isolation and characterization of ES cells with both FGF-4 alleles inactivated.

Binding of transcription factors to widely-separated cis-regulatory elements of the murine FGF-4 gene.

Embryonal carcinoma (EC) cells and their embryo-derived counterparts, embryonic stem (ES) cells, have been used extensively to study the transcriptional regulation of the fibroblast growth factor-4 (FGF-4) gene. The FGF-4 gene is expressed in EC cells and ES cells, but it is repressed in their retinoic acid (RA)-induced differentiated counterparts. Previous studies have shown that the transcription of the FGF-4 gene is controlled by cis-regulatory elements located in the 5' flanking region of the gene, and by a powerful enhancer located approximately 3 kb downstream from the transcription start site.

Effects of oxidation and reduction on the binding of transcription factors to cis-regulatory elements located in the FGF-4 gene.

Previous studies have shown that the addition of reducing agents to the culture medium of embryonic cell lines stimulates their growth. Moreover, recent studies have shown that the redox state of several transcription factors affects their binding to DNA. In light of these findings, we employed gel mobility shift analysis to examine the effects of oxidation and reduction on the ability of transcription factors to bind cis-regulatory elements located in the FGF-4 gene, which is expressed during early mammalian development.

Cl- secretion by trachea of CFTR (+/-) and (-/-) fetal mouse.

The absence of pathologic changes in newborn cystic fibrosis (CF) lung suggests that the fetal CF lung is inflated with a normal volume of liquid and that Cl- is secreted through paths other than the cystic fibrosis transmembrane conductance regulator (CFTR)-associated Cl- channel. We studied liquid content of distal lung and transepithelial electrical potential difference (PD) of cultured cystic tracheal explants from 16 to 19 day gestation fetal mice of CFTR (+/-)(heterozygous) females that were mated with CFTR (-/-) "knockout" males. Distal lung water content was not affected by fetal genotype.

A murine model of cystic fibrosis.

We have generated a mouse line in which the cystic fibrosis transmembrane conductance regulator (CFTR) gene has been mutated by gene targeting. Like human cystic fibrosis (CF) patients, mice lacking a functional CFTR gene, referred to as CFTR(-/-) mice, show increased numbers of goblet cells and obstruction of glands with inspissated eosinophilic secretions. The obstruction of glands often results in the destruction of gland-containing tissues in these animals.

Cystic fibrosis heterozygote resistance to cholera toxin in the cystic fibrosis mouse model.

The effect of the number of cystic fibrosis (CF) alleles on cholera toxin (CT)-induced intestinal secretion was examined in the CF mouse model. CF mice that expressed no CF transmembrane conductance regulator (CFTR) protein did not secrete fluid in response to CT. Heterozygotes expressed 50 percent of the normal amount of CFTR protein in the intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion in intestinal epithelium and secreted 50 percent of the normal fluid and chloride ion and fluid secretion suggests that CF heterozygotes might possess a selective advantage of resistance to cholera.

Abolition of anaphylaxis by targeted disruption of the high affinity immunoglobulin E receptor alpha chain gene.

Mast cells and basophils, which are activated by immunoglobulin E (IgE) and allergen, play a prominent role in anaphylaxis. However, they express at least three types of IgE receptor, including the high affinity IgE receptor (Fc epsilon RI). The relative contribution of these IgE receptors, and possibly other receptors such as Fc epsilon RII/CD23 and Mac-2, to the genesis of in vivo anaphylaxis is still unclear.

An animal model for cystic fibrosis made by gene targeting.

Cystic fibrosis results from defects in the gene encoding a cyclic adenosine monophosphate-dependent chloride ion channel known as the cystic fibrosis transmembrane conductance regulator (CFTR). To create an animal model for cystic fibrosis, mice were generated from embryonic stem cells in which the CFTR gene was disrupted by gene targeting. Mice homozygous for the disrupted gene display many features common to young human cystic fibrosis patients, including failure to thrive, meconium ileus, alteration of mucous and serous glands, and obstruction of glandlike structures with inspissated eosinophilic material.

LCMV-specific, class II-restricted cytotoxic T cells in beta 2-microglobulin-deficient mice.

Intracranial infection of normal mice with lymphocytic choriomeningitis virus (LCMV) causes meningitis and death mediated by CD8+ major histocompatibility complex (MHC) class I-restricted cytotoxic T lymphocytes (CTLs). beta 2-Microglobulin-deficient mice (beta 2M-/-) do not express functional MHC class I proteins and do not produce significant numbers of CD8+ T cells. When beta 2M-/- mice were infected with LCMV, many died from LCMV disease and produced a specific response to LCMV mediated by CD4+ CTLs that were class II-restricted.

Toward an animal model of cystic fibrosis: targeted interruption of exon 10 of the cystic fibrosis transmembrane regulator gene in embryonic stem cells.

A gene-targeting construct was made containing 7.8 kilobases of DNA spanning exon 10 of the mouse cystic fibrosis transmembrane regulator (CFTR) gene in which part of the exon has been replaced by two neomycin-resistance (Neo) genes driven by different promoters. (This replacement introduces a chain-termination codon at amino acid position 489 in the CFTR sequence).