Ch. 22
Ch. 22. Experimental Systems
In vivo vs. in vitro systems
Experimental animals
Inbred strains reduce variation
Adoptive transfer expts.
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Cell culture systems
Primary lymphoid cell cultures are derived from blood or lymphoid organs.
Cloned lymphoid cell lines are important tools.
Hybrid lymphoid cell linesMonoclonal Ab’sT cell hybridomas, representing Th and Tc
lineages
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p. 550
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Protein biochemistry
Radiolabeling techniques - sensitivity
Biotin labels combine with avidin
Gel electrophoresis – proteins separated bysize and charge
X-ray crystallography – structural information
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Recombinant DNA technology
Restriction enzymes cleave DNA at precise
sequences.
DNA sequences are cloned into vectors, wherein DNA is replicated.
Some vectors: bacterial and insect viruses, retroviruses, plasmids
DNA clones are selected by hybridization.
Ch. 22
p. 556
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p. 557
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Southern blotting detects DNA of a given sequence.
Northern blotting detects mRNA.
The polymerase chain reaction (PCR) amplifies small amounts of DNA.
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p. 558
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p. 559
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Gene transfer into mammalian cells
Cloned genes transferred into cultured cells allow in vitro analysis of gene function.
Cloned genes transferred into mouse embryos allow in vivo analysis of gene function.
In knockout mice, targeted genes are disrupted.
“Knock-in” technology allows the replacement of an endogenous gene.
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p. 562
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p. 563
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Microarrays – An approach for analyzing gene expression
“Expression profiling”
Some are composed of cDNA
Some are composed of oligonucleotides (“oligos”)
Good diagnostic tool for some diseases
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p. 569
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p. 570
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