Transformation and Immortalization of Cultured Cells PDF

Transformation and Immortalization of cultured cells  Transformation of cultured cells implies a spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression. Transformation is associated with genetic instability and three major classes o...

Transformation and Immortalization of cultured cells  Transformation of cultured cells implies a spontaneous or induced permanent phenotypic change resulting from a heritable change in DNA and gene expression. Transformation is associated with genetic instability and three major classes of phenotypic change, one or all of which may be expressed in one cell strain: 1. Immortalization, the acquisition of an infinite life span, 2. Aberrant growth control, the loss of contact inhibition of cell motility, density limitation of cell proliferation, and anchorage dependence, and 3. Malignancy, as evidenced by the growth of invasive tumors in vivo. The term transformation is used here to imply all three of these processes. Immortalization What is the need? Immortalized cell lines are either tumorous cells that do not stop dividing or cells that have been artificially manipulated to proliferate indefinitely and can thus be cultured over several generations. Because immortalized cells continuously divide, they eventually fill up the dish or flask in which they grow. By passaging (also known as splitting), scientists transfer a fraction of the multiplying cells into new dishes to provide space for continuing proliferation. Common immortalized cell lines include human embryonic kidney 293T (HEK-293T) cells and HeLa cells. Immortalization: advantages and disadvantages Well characterized Cannot be considered normal Homogeneous, genetically identical cells as in in vivo conditions populations of cells, allowing for After several passages, cell consistent and reproducible results. characteristics can change and Easier to culture than cells used in become even more different from primary cultures those of a normal cell. Grow quickly and continuously Advantag Disadvanta es ges Immortalization with Viral Genes The SV40 LT gene is often used to induce immortalization. The product of this gene, T antigen, is known to bind p53 which overrides cell cycle. By doing so, it not only allows an extended proliferative life span but also restricts the DNA surveillance activity of genes like p53, thereby allowing an increase in genomic instability and an increased chance of generating further mutations favorable to immortalization (e.g., the upregulation of telomerase or the downregulation of one of the telomerase inhibitors).