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 cells Many 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