Chapter 6: MANIPULATING CELLS AND VIRUSES

IN CULTURE

Reasons for using cultured cells:

∑ Reduce complexity by manipulating a single cell type.

∑ Easy to control experimental conditions.

∑ Clonal growth insures genetic homogeneity.

Microorganisms:

∑ E. coli and yeast are model prokaryotic and eukaryotic microorganisms, respectively; Can be grown on minimal media (T6-1); Can be grown from single cells on agar plates (6-1).

∑ Replica plating can be used to test growth on different media; Genetic selection for auxotrophic mutants (6-2).

Cultured animal cells:

∑ Nutritional requirements (amino acids, vitamins, salts, glucose serum) for growth of animal cells (T6-2); Cells are normally in contact with other cells and the extracellular matrix in intact animals; Common components of the extracellular matrix; Cultured cells grow as colonies on a surface on which they secrete extracellular matrix components (6-3).

∑ Animal tissues or embryos dissociated by proteases and low Ca⁺² can be cultured as primary cell cultures; Specialized cells types (muscles, neurons, etc.) are relatively difficult to culture, whereas fibroblasts and epithelial cells are easier to culture; Types of epithelia (6-4); Primary cultures have a limited culture life and are called cell strains (6-5a).

∑ Some cultured cells undergo spontaneous genetic changes (i.e. oncogenic transformation) that enables growth indefinitely and are called immortalized cell lines; Tranformation occurs rarely in human cell culture, but routinely in rodent cell culture (6-5b); Many cultured cell lines have chromosomal abnormalities; Most cell lines are undifferentiated, some cell lines can carry out functions of differentiated cells (6-7).

∑ Cutured animal cells can fuse to form heterokaryons; Can use cultured cells to identify genes through formation of heterokaryons; This has been done with human-mouse hybrids, and panels of hybrid cells exist that have all mouse chromosomes and variable numbers of human chromosomes; Human-mouse hybrid panels enable mapping of human genes to certain chromosomes.

∑ Use of the purine and pyrimidine nucleotide synthetic pathways to select hybrid cells; Antifolates block de novo synthesis of nucleotides; Function of the TK and HGPRT enzymes (6-9); Use of HAT media to select for hybrid cells.

∑ B lymphocytes, epitopes and monoclonal entibodies; Fusing transformed lymphocyte cells (myelomas) with normal lymphocytes can generate hybridomas that make a single monoclonal antibody; Procedure for generating monoclonal antibodies (6-10).

Viruses:

∑ Helical and icosahedral capsid structures of animal viruses (6-11); Detecting and quantifying viruses using plaque assays (6-14).

∑ Classification of viruses based on genome composition and pathway of mRNA formation (6-20); Properties of different viral classes (T6-3); Retroviruses (6-22); Use of viruses as gene delivery systems for gene therapy.