Generation and characterization of p53 mutant mice.

p53 is one of the most well-characterized members of the tumor suppressor gene family. The role of p53 in controlling cellular homeostasis has proven critical, with over half of all human tumors having either lost or mutated p53. The emergence of technology facilitating the ablation of a gene within an animal's genome allowed great advances in the study of p53. The p53 knockout mouse was one of the first of its kind and provided a powerful tool for the study of p53. Production of the p53 knock-out mouse demonstrated the protein's dispensability during embryogenesis, while highlighting its essential role in controlling tumor formation. A variety of p53 mutant models have emerged since the original p53 knock-out mouse, along with improved techniques for regulating gene targeting. This chapter describes the necessary steps and protocols involved in producing a mutant mouse as well as the characterization that follows.

Generation and characterization of p53 null transformed hepatic progenitor cells: oval cells give rise to hepatocellular carcinoma.

Oval cells are bipotential liver stem cells able to differentiate into hepatocytes and bile duct epithelia. In normal adult liver oval cells are quiescent, existing in low numbers around the periportal region, and proliferate following severe, prolonged liver trauma. There is evidence implicating oval cells in the development of hepatocellular carcinoma, and hence the availability of an immortalized oval cell line would be invaluable for the study of liver cell lineage differentiation and carcinogenesis. A novel approach in the generation of cell lines is the use of the p53 knockout mouse. Absence of p53 allows a cell to cycle past the normal Hayflick limit, rendering it immortalized, although subsequent genetic alterations are thought necessary for transformation. p53 knockout mice were fed a choline-deficient, ethionine-supplemented diet, previously shown to increase oval cell numbers in wild-type mice. The oval cells were isolated by centrifugal elutriation and maintained in culture. Colonies of hepatic cells were isolated and characterized with respect to phenotype, growth characteristics and tumorigenicity. Analysis of gene expression by Northern blotting and immunocytochemistry suggests they are oval-like cells by virtue of albumin and transferrin expression, as well as the oval cell markers alpha fetoprotein, M(2)-pyruvate kinase and A6. Injection into athymic nude mice shows the cell lines are capable of forming tumors which phenotypically resemble hepatocellular carcinoma. Thus, the use of p53 null hepatic cells successfully generated immortalized and tumorigenic hepatic stem cell lines. The results presented support the idea that deleting p53 allows immortalization and contributes to the transformation of the oval-like cell lines. Further, the tumorigenic status of the cell lines is direct evidence for the participation of oval cells in the formation of hepatocellular carcinoma.