Synthesis of a mouse model of the dysfibrinogen Vlissingen/Frankfurt IV.

The dysfibrinogen Vlissingen/Frankfurt IV is characterized as a deletion of Asn319 and Asp320 from the C-terminus of the gamma-chain of fibrinogen. This dysfibrinogen, which was identified in several family members that are all heterozygous for the in-frame 6-bp deletion, is associated with both venous and arterial thrombosis. Here, we describe the generation of a murine model of the V/F IV dysfibrinogen using gene targeting of mouse gamma-chain DNA. Preliminary analysis shows that the human and mouse variant fibrinogens are similar: analogous to the human V/F IV protein, the D1 fragment of the variant mouse fibrinogen is partially protected from digestion in the presence of calcium or Gly-Pro-Arg-Pro. These heterozygous mice provide the first opportunity to examine the association of thrombophilia and dysfibrinogenemia in a controlled genetic background.

Targeted disruption of the mouse topoisomerase I gene by camptothecin selection.

Topoisomerase I has ubiquitous roles in important cellular functions such as replication, transcription, and recombination. In order to further characterize this enzyme in vivo, we have used gene targeting to inactivate the mouse Top-1 gene. A selection protocol using the topoisomerase I inhibitor camptothecin facilitated isolation of embryonic stem cell clones containing an inactivated allele; isolation of correctly targeted clones was enhanced 75-fold over that achieved by normal selection procedures. The disrupted Top-1 allele is embryonic lethal when homozygous, and development of such embryos fails between the 4- and 16-cell stages. Both sperm and oocytes containing the inactive allele maintain viability through the fertilization point, and thus gene expression of topoisomerase I is not required for gamete viability. These studies demonstrate that topoisomerase I is essential for cell growth and division in vivo. The Top-1 gene was also shown to be linked to the agouti locus.

Hepatocellular and hepatic peroxisomal alterations in mice with a disrupted peroxisomal fatty acyl-coenzyme A oxidase gene.

Peroxisomal genetic disorders, such as Zellweger syndrome, are characterized by defects in one or more enzymes involved in the peroxisomal beta-oxidation of very long chain fatty acids and are associated with defective peroxisomal biogenesis. The biologic role of peroxisomal beta-oxidation system, which consists of three enzymes: fatty acyl-CoA oxidase (ACOX), enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), and thiolase, has been examined in mice by disrupting ACOX gene, which encodes the first and rate-limiting enzyme of this system. Homozygous (ACOX -/-) mice lacked the expression of ACOX protein and accumulate very long chain fatty acids in blood. However, these homozygous mice are viable, but growth-retarded and infertile. During the first 3-4 months of age, the livers of ACOX -/- mice reveal severe microvesicular fatty metamorphosis of hepatocytes. In such steatotic cells, peroxisome assembly is markedly defective; as a result, they contain few or no peroxisomes. Few hepatocytes in 1-3-month-old ACOX -/- mice contain numerous peroxisomes, and these peroxisome-rich hepatocytes show no fatty change. At this stage, the basal mRNA levels of HD, thiolase, and other peroxisome proliferator-induced target genes were elevated in ACOX -/- mouse liver, but these mice, when treated with a peroxisome proliferator, showed no increases in the number of hepatic peroxisomes and in the mRNAs levels of these target genes. Between 4 and 5 months of age, severe steatosis resulted in scattered cell death, steatohepatitis, formation of lipogranulomas, and focal hepatocellular regeneration. In 6-7-month-old animals, the newly emerging hepatocytes, which progressively replaced steatotic cells, revealed spontaneous peroxisome proliferation. These livers showed marked increases in the mRNA levels of the remaining two genes of the beta-oxidation system, suggesting that ACOX gene disruption leads to increased endogenous ligand-mediated transcription levels. These observations demonstrate links among peroxisomal beta-oxidation, development of severe microvesicular fatty liver, peroxisome assembly, cell death, and cell proliferation in liver.

Single-copy transgenic mice with chosen-site integration.

We describe a general way of introducing transgenes into the mouse germ line for comparing different sequences without the complications of variation in copy number and insertion site. The method uses homologous recombination in embryonic stem (ES) cells to generate mice having a single copy of a transgene integrated into a chosen location in the genome. To test the method, a single copy murine bcl-2 cDNA driven by either a chicken beta-actin promoter or a human beta-actin promoter has been inserted immediately 5' to the X-linked hypoxanthine phosphoribosyltransferase locus by a directly selectable homologous recombination event. The level of expression of the targeted bcl-2 transgene in ES cells is identical in independently isolated homologous recombinants having the same promoter yet varies between the different promoters. In contrast, the expression of bcl-2 transgenes having the same (chicken beta-actin) promoter varies drastically when they are independently integrated at random insertion sites. Both promoters direct broad expression of the single-copy transgene in mice derived from the respective targeted ES cells. In vitro and in vivo, the human beta-actin promoter consistently directed a higher level of transgene expression than the chicken beta-actin promoter.

Prostaglandin synthase 2 gene disruption causes severe renal pathology in the mouse.

The prostaglandin endoperoxide H synthase isoform 2, cyclooxygenase 2 (COX-2), is induced at high levels in migratory and other responding cells by pro-inflammatory stimuli. COX-2 is generally considered to be a mediator of inflammation. Its isoform, COX-1, is constitutively expressed in most tissues and is thought to mediate "housekeeping" functions. These two enzymes are therapeutic targets of the widely used nonsteroidal anti-inflammatory drugs (NSAIDs). To investigate further the different physiologic roles of these isoforms, we have used homologous recombination to disrupt the mouse gene encoding COX-2 (Ptgs2). Mice lacking COX-2 have normal inflammatory responses to treatments with tetradecanoyl phorbol acetate or with arachidonic acid. However, they develop severe nephropathy and are susceptible to peritonitis.

Prostaglandin synthase 1 gene disruption in mice reduces arachidonic acid-induced inflammation and indomethacin-induced gastric ulceration.

Cyclooxygenases 1 and 2 (COX-1 and COX-2) are key enzymes in prostaglandin biosynthesis and the target enzymes for the widely used nonsteroidal anti-inflammatory drugs. To study the physiological roles of the individual isoforms, we have disrupted the mouse Ptgs1 gene encoding COX-1. Homozygous Ptgs1 mutant mice survive well, have no gastric pathology, and show less indomethacin-induced gastric ulceration than wild-type mice, even though their gastric prostaglandin E2 levels are about 1% of wild type. The homozygous mutant mice have reduced platelet aggregation and a decreased inflammatory response to arachidonic acid, but not to tetradecanoyl phorbol acetate. Ptgs1 homozygous mutant females mated to homozygous mutant males produce few live offspring. COX-1-deficient mice provide a useful model to distinguish the physiological roles of COX-1 and COX-2.

Genetic control of blood pressure and the angiotensinogen locus.

Variants of the human angiotensinogen gene have been linked in some studies to increased circulating angiotensinogen levels and essential hypertension. To test for direct causality between genotypes at the angiotensinogen locus and blood pressures, we have studied mice carrying zero, one, two, three, or four functional copies of the murine wild-type angiotensinogen gene (Agt) at its normal chromosomal location. Plasma angiotensinogen levels increase progressively, although not linearly, from zero in the zero-copy animals to 145% of normal in the four-copy animals. Mice of all genotypes are normal at birth, but most zero-copy animals die before weaning. The kidneys of the zero-copy animals show pathological changes as adults, but the kidneys are normal in the other genotypes. One adult zero-copy male tested was fertile. The blood pressures of the one-copy through four-copy animals show significant and almost linear increases of approximately 8 mmHg per gene copy despite their normal compensatory mechanisms being intact. These results establish a direct causal relationship between Agt genotypes and blood pressures.