chapter 6 manipulating cells in culture. advantages of working with cultured cells over intact...
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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