Chapter 6 Manipulating Cells in Culture

Advantages of working with cultured cells over intact organisms More homogeneous than cells in tissues Can control experimental conditions Can isolate single cells to grow into a colony of genetically homogeneous clone cells

Growth of microorganisms in culture Examples: E. coli and the yeast S. cerevisiae Have rapid growth rate and simple nutritional requirements Can be grown on semisolid agar Mutant strains can be isolated by replica plating Yeast colonies

Growth of microorganisms in culture

Replica plating

Growth of animal cells in culture Requires rich media including essential amino acids, vitamins, salts, glucose, and serum Most grow only on special solid surfaces A single mouse cell Figure 6-36 A colony of human cellsMany colonies in a petri dish

Growth of animal cells in culture

Primary cells and cell lines Primary cell cultures are established from animal tissues Most cells removed from an animal grow and divide for a limited period of time (about 50 doublings), then eventually die Certain “transformed cells” may arise that are immortal and can be used to form a cell line Transformed cells may be derived from tumors or may arise spontaneously

Establishment of a cell culture Figure 6-37

Cell fusion Two different cells can be induced to fuse thereby creating a hybrid cell (heterokaryon) Interspecific hybrids may be used for somatic-cell genetics Certain hybrid cells (hybridomas) are used to produce monoclonal antibodies

De Novo and salvage pathways for nucleotide synthesis Figure 6-9

Figure 6-10 Producing a monoclonal antibody to protein X

Chapter 5.5 Purification of cells and their parts

Figure 5-34 Purification of specific cells by flow cytometry Requires fluorescent tag for desired cell

Example: FACS data Figure 5-35

Purification of cell parts Understanding the roles of each each cell component depends on methods to break open (lyse) cells and separate cell components for analysis Cell lysis is accomplished by various techniques: blender, sonication, tissue homogenizer, hypotonic solution Separation of cell components generally involves centrifugation

Cell fractionation by differential centrifugation Figure 5-36

Organelle separation by equilibrium density-gradient centrifugation Figure 5-37