Transgenic and gene disruption techniques in the study of neurocarcinogenesis

Abstract

Transgenic technologies have come of age, and the field of carcinogenesis has profited extensively from the availability of these methods. Both the inappropriate expression of dominant oncogenes in specific tissues and the ability to "knock out" tumor suppressor genes in mammalian organisms have enabled substantial advancements of our understanding of development and progression of the neoplastic phenotype. In the first part of this article, we review the most popular techniques for modification of the mammalian genome in vivo, i.e. microinjection of fertilized eggs, retrovirus-mediated gene transfer, and targeted gene deletion through homologous recombination. Subsequently, we attempt a critical evaluation of the available models of neurocarcinogenesis, and discuss their impact and future potential for the study of cancer in the nervous system.

Noninvasive Assessment of E2F-1–Mediated Transcriptional Regulation In vivo

“…After separation (1 h; 100V) proteins were blotted to a Hybond-ECL membrane (Amersham Pharmacia Biotech) in standard Trisglycin transfer buffer (100V; 240 mA; 1.25 h). After blocking nonspecific binding (5% nonfat dry milk in 0.1% PBS-Tween20; 1 h), the membrane was washed (0.1% PBS-Tween20) and then probed with primary antibodies anti-E2F KH95 (1:500 dilution), anti-actin C-2 (1:200 dilution; Santa Cruz), respectively, in 5% dry milk and 0.1% PBS-Tween20 (22). After washing, secondary antibodies Goat Anti-Mouse IgG with horseradish peroxides (Southern Biotechnology Associates, Inc.; 1:10,000) were conjugated in 5% dry milk in 0.1% PBS Tween20 for 1 h, and for final protein detection, chemiluminescent reaction (Pierce Biotechnology, Inc.) were used.…”

“…Cells were permeabilized (0.2% Triton X-100 in PBS; ref. 22) and washed with PBS (3 Â 5 min). Unspecific binding of antibodies was prevented by blocking cells [3% bovine serum albumin (BSA); 60 min].…”

“…Cellular DNA was stained with propidium iodide (SIGMA-ALDRICH Chemie GmbH; ref. 22). The cellular DNA content was analyzed by flow cytometry ( fluorescence-activated cell sorting; FACSCalibur; Becton Dickinson).…”

Of mice and men: the evolution of animal welfare guidelines for cancer research

“…Following one of the tenets of the 3Rs (Russell and Burch, 1959), refinement, several laboratories have developed genetically and histologically accurate models of carcinoma by gain-of-function (transgenic) approaches and targeted deletion (including tissue-specific) strategies. Transgenic mice express the gene of interest in all cells *Correspondence: Dr B Leyland-Jones; E-mail: Leyland@emory.edu (Aguzzi et al, 1995), whereas knockout mice lose expression of the targeted gene in all cells that would normally express it (Macleod and Jacks, 1999). These are gene-manipulated 'extra-evolutionary species' of mice bred for medical/scientific purposes.…”

Tracking prions: the neurografting approach

“…The complexity of these questions, along with our limited understanding of the nature of the infectious agent, suggests that it may be very difficult to devise suitable systems to address them experimentally. However, the recent generation of genetic in vivo model systems, such as transgenic and knockout mice [7], has opened new, promising avenues of investigation. In particular, the experiments described below were made possible by the availability of mice expressing, at various levels, normal and mutated forms of the prion protein, as well as knockout mice which bear hetero-or homozygous ablations of the Prnp gene which encodes the prion protein.…”