In Vitro Experiments

Representitive organisms - know lots about so can study them and apply them

• Bacteria - E.coli
• Unicellular Eukaryotes - Yeast S.cerevisae (budding reproduction)
  
• Simple multicellular - nematode worm - C.elegans (neuroscience model)
• Insect - fruit fly - D.melanogaster (genetics - fast mutation)
• Fish - zebra danio - Danio rerio (transparent eggs - embryonic development)
• Amphibian - african clawed frog - X.laevis (large single celled eggs - inject genes)
• Mammal - mouse
• Plant - Arabidopsis thaliana

Variation of organs in people

Statistal analysis - cope with variation (especially in wild pop.)

Labs - genetically identical organisms (lab strains)

Cell lines - cell culture developed from 1 cell = uniform genetic makeup

• Does not remove morphological differences

• Well-characterised, near indentical organisms
• Outbred - some genetic variation (interbreeding)
• Inbred e.g. mice
  • Produced from 20+ brother/sister matings = 98% genetically identical
  • 40 matings = 99.5% genetically identical
  • Rattus norvengious strains:
    • Wistar (lab white rat) - orginal colony from Wistar institute in 1906
    • Sprague-Dawley - Wistar rat and mystery male

Fatty Zucker rat - obese rate - determine obesity gene

Biobreeding Diabetes Prone (BBDP) rat - diabetes cure

Fruit flies - expose to radiation to mutate e.g. alcohol sensitivity (barfly, tipsy)

Removing/silencing gene - detemines importance

C57 block/6 (mouse genome) -

• Stargazer - epilepsy
• Muscular dystrophy
• Rheumatoid arthritis

Sanger institute has around 3000 knockout mice

Knock-ins - Oncomouse (cancer mouse)

E.coli - standard genetic manipulation tool

• Easy culture
• Easy harvest
• Rapid population growth
• Metabollicaly handicapped - can't grow in 'wild' = rely on food
• Antibiotic resistant - kill off contaminated strains

Natural or genetically engineered

Grown in liquid (broth - nutrient rich) or solid (agar plates - gel) mediums

• Frozen bacteria stock thawed 
• Heat-sterilised metal loop dipped in stock
• Streak plate = aseptic technique
• Each new pattern dilutes bacteria
• Agar plate inverted and incubated - 37'C
• Cell replicate = visible colonies
• Colony = genetically identical cells
• Colony scrapped off -> broth
• Grow up in bulk

• Easy to observe
• Easy to manipulate - no barriers
• More pure (technically - contamination occurs)

Adherent monolayers at bottom of flask

Contain growth media e.g. DMEM

Red pH-sensitive pigment - detect acidity changes

Usually supplemented with fetal calf serum and growth factors

37'C 

Issues:

• Monolayers - cant invesigate 3D structure effect etc
• Limited life
• Replicating cell type required for continued culture

• Stem cells - can be differentiated by chemical treatment
• Retinoic acid = fibroblast
• Petri dish + retinoic acid = neurons/glia
• Petri dish + DMSO = cardiac and skeletal muscle

• Triculture on petri dish scaffold
  • Human hepatocytes
  • Human endothelial cells
  • Mouse fibroblasts (connective tissue)
• 'Liver' transplanted into mouse
• Intergrates with circulatory system
• Human liver proteins releases
• Proteins extracted and used

Organotypic tissue culture - structure and function preserved (thin slices)

• Hard to keep alive - blood supply gone