Ch. 22

Ch. 22. Experimental Systems

In vivo vs. in vitro systems

Experimental animals

Inbred strains reduce variation

Adoptive transfer expts.

Ch. 22p. 546

Ch. 22

p. 547

Ch. 22

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

Ch. 22p. 548

Ch. 22

Ch. 22

p. 550

Ch. 22

Ch. 22

Ch. 22

Protein biochemistry

Radiolabeling techniques - sensitivity

Biotin labels combine with avidin

Gel electrophoresis – proteins separated bysize and charge

X-ray crystallography – structural information

Ch. 22

p. 551

Ch. 22

p. 552

Ch. 22p. 552

Ch. 22

Ch. 22

Ch. 22

p. 553

Ch. 22

p. 555

Ch. 22

Ch. 22

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

Ch. 22

p. 557

Ch. 22

Ch. 22

Ch. 22

p. 557

Ch. 22

Ch. 22

Ch. 22p. 558

Ch. 22

Southern blotting detects DNA of a given sequence.

Northern blotting detects mRNA.

The polymerase chain reaction (PCR) amplifies small amounts of DNA.

Ch. 22

p. 558

Ch. 22

p. 559

Ch. 22 p. 560

Ch. 22

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.

Ch. 22

p. 562

Ch. 22

p. 563

Ch. 22

Ch. 22

Ch. 22 p. 564

Ch. 22

p. 565

Ch. 22

Ch. 22

p. 566

Ch. 22

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

Ch. 22

p. 569

Ch. 22

p. 570