Transcript Chapter 4

Chapter 6
Manipulating Cells in Culture
Advantages of working with cultured cells over intact
organisms
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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
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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
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Requires rich media including essential amino acids,
vitamins, salts, glucose, and serum
Most grow only on special solid surfaces
A single mouse cell
A colony of human cells
Many colonies in a petri dish
Figure 6-36
Growth of animal cells in culture
Primary cells and cell lines
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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
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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
Producing a monoclonal
antibody to protein X
Figure 6-10
Chapter 5.5 Purification of cells and their parts
Purification of specific
cells by flow cytometry
Requires fluorescent
tag for desired cell
Figure 5-34
Example: FACS data
Figure 5-35
Purification of cell parts
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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