Cell and Animal Models for Studying Basic Cell Biology and Human Disease

Model Systems in Biology

Learning Outcomes

  • Understand the range of model systems used in biological research and the reasons for their selection.
  • Recognize how animal and cellular models enable experimental testing of fundamental cell biology concepts and disease mechanisms, linking this knowledge to other lectures.
  • Understand the types of genetic manipulation suited for different model organisms (from the associated screencast).

Why Study Model Systems?

  • Animal and cellular models offer opportunities for experimental testing of basic cell biology and disease mechanisms.
  • These models often serve as the "standard" in vivo approach in biomedical investigations.
  • It is important to consider whether these models accurately represent the phenomena under investigation.

Model Organisms in Biology

  • Model organisms are species developed over time for scientific inquiry.
  • Essential characteristics include ease of handling in the laboratory.
  • Other factors considered include cost, size, lifespan, and availability of a sequenced genome.
  • Model species often represent important taxonomical properties, with the goal that biochemical findings are broadly relevant to other species.

Examples of Model Organisms:

  • E. coli
  • Yeasts
  • Flies (Drosophila)
  • Worms (Caenorhabditis elegans)
  • Fish (Danio rerio) – Zebrafish
  • Frog/Toad (Xenopus)
  • Mice
  • Pigs and non-human primates
  • Cells in culture
  • Arabidopsis – a model plant

Escherichia coli

  • E. coli is one of the oldest and most studied model organisms.
  • It is not particularly useful for understanding eukaryote biology.

Advantages and Disadvantages of Different Models

  • Key parameters to consider when choosing a model organism include:
    • Cell volume (V)
    • Cell length (L)
    • Doubling time (t)
    • Water content
    • Membrane composition
    • Protein content
    • mRNA content
    • Lipid content
    • Inorganic ion content
    • Ribosome count
    • DNA base pairs (bp)

Data for different cell types:

  • (A) Bacterial cell (E. coli):

    • V ≈ 1 \, µm^3
    • L ≈ 1 \, µm
    • t ≈ 1 \, hour
    • Protein: 5 × 10^5
    • mRNA: 10^8
    • Lipid: 2 × 10^{10}
    • Inorganic ions: 3 × 10^6
    • Ribosomes: 2 × 10^3
    • DNA: 5 × 10^6 \, bp

  • (B) Yeast cell (S. cerevisiae):

    • V ≈ 30 \, µm^3
    • L ≈ 5 \, µm
    • t ≈ 3 \, hours
    • Protein: 10^7
    • mRNA: 10^9
    • Lipid: 6 × 10^{11}
    • Inorganic ions: 3 × 10^9
    • Ribosomes: 10^8
    • DNA: 1.2 × 10^7 \, bp

  • (C) Mammalian cell (HeLa):

    • V ≈ 3000 \, µm^3
    • L ≈ 20 \, µm
    • t ≈ 1 \, day
    • Protein: 10^9
    • mRNA: 6 × 10^{13}
    • Lipid: 10^{11}
    • Inorganic ions: 2 × 10^{11}
    • Ribosomes: 2 × 10^5
    • DNA: 3 × 10^9 \, bp

Yeast as a Minimal Model Eukaryote

  • Saccharomyces cerevisiae
  • Schizosaccharomyces pombe
  • Unicellular fungi used in the production of wine, beer, and bread.
  • Under optimal laboratory conditions, they can double every 90-100 minutes.
  • They have both diploid and haploid life stages.

Yeast: Saccharomyces cerevisiae Life Cycle

  • The yeast life cycle includes both haploid (a) and diploid stages.

Applications of Yeast Models

Yeast models are valuable in studying:

  • Protein folding, quality control, and degradation
  • Lipid biology
  • Vesicular trafficking and fusion
  • Mitochondria and oxidative stress
  • Lysosomal and peroxisomal function
  • Autophagy
  • Apoptosis
  • Cell cycle
  • Chaperone proteins (e.g. Hsps)
  • Protein-remodelling factors (e.g. Hsp104)
  • Osmolytes (e.g. trehalose)
  • Proteolytic machineries (e.g. UPS, lysosomal and autophagic mechanisms)

Random Chemical Mutation Approaches

  • Forward Genetics: Mutagenized culture -> replica plate -> Screen for phenotype and then clone the gene mutated. -> Gene identification (Complementation)
    • Example: glycolysis mutant with ethanol as a carbon source instead of glucose.
  • Forward Chemical Genetics: Chemical library -> replica plate -> Screen for phenotype -> Target identification (Chemogenomic profiling)
    • Example: glycolysis inhibitor

Learning About Secretion From Yeast

  • Yeast sec mutants, temperature-sensitive for protein secretion, define five steps in secretory protein maturation.
  • Class A: Accumulation in the cytosol; defective transport into the ER.
  • Class B: Accumulation in rough ER; defective budding of vesicles from the rough ER.
  • Class C: Accumulation in ER-to-Golgi transport vesicles; defective fusion of transport vesicles with Golgi.
  • Class D: Accumulation in Golgi; defective transport from Golgi to secretory vesicles.
  • Class E: Accumulation in secretory vesicles; defective transport from secretory vesicles to the cell surface.

Caenorhabditis elegans - a Nematode Worm

  • A simple model for multi-cellular organisms
  • Size of adult worm ~1 mm
  • Life cycle:
    • Embryonic development (14 hr)
    • L1 larva (12 hr)
    • L2 larva (13 hr)
    • Predauer (L2d) when there is crowding and low food
    • L3 larva (8 hr)
    • Dauer larva (several months)
    • L4 larva (8 hr)
    • Young adult (10 hr)
    • Adult (8 hr)

Caenorhabditis elegans – Cell Number

  • C. elegans has a fixed number of somatic cells.
  • 1031 in male
  • 959 in the hermaphrodite.
  • The lineage of every cell in the adult has been traced.

Caenorhabditis elegans – Connectome

  • C. elegans is the only animal for which a complete wiring diagram – the connectome – of the nervous system has been determined by electron microscopy.

Drosophila melanogaster

  • Drosophila melanogaster is another popular organism sharing many properties with C. elegans.
  • Easy to breed and small, allowing experiments on thousands of individuals.

Studies in Drosophila

  • Studies in Drosophila provide a key to vertebrate development.
  • The life cycle takes ~7 - 10 days.
  • Arguably the most important developmental model for multicellular organisms – Hox genes discovered in this organisms.

Zebrafish

Zebrafish Danio rerio

  • Transparent embryos which develop outside the mother.
  • Can withstand higher doses of mutagens, allowing the creation of more mutants.
  • Their cells and body parts can regenerate.

Zebrafish Biology and Applications

  • Biology: Embryo, Larvae, Adult
  • Functional genomics
    • Forward genetics: Large scale mutagenesis (and positional cloning): Chemical, Insertional (virus and transponson), Inbreeding
    • Reverse genetics: Germline transgenesis (overexpression/ dominant negative/reporter systems): Plasmid transgenes, transposon, virus, Somatic transgenesis (knock down): Morpholinos, ribozymes, siRNA, PNA, Site directed mutagenesis: TILLING
  • Expression profiling: Microarray: cDNA, oligonucleotide, SNP, miRNA. Proteomics: Protein arrays. Other techniques: rtPCR and in situ hybridization techniques
  • Bioinformatics: Comparative genomics, Homology/conservation, In silico cloning, Electronic expression comparison
  • Applications
    • Human biomedicine: Phenotypic (disease) models: Inherited diseases, infectious diseases, regenerative and tissue repair models. High throughput screening (in vivo): Drug screening, toxicology
    • Aquatic biomedicine: Phenotypic (disease) models: Inherited and infectious diseases, Immunity: DNA vaccine production
    • "Aqua" biotechnology: Bioreactors (recombinant protein production): Human coagulation factor (hFVII), Genetic improvements in aquaculture: Pet fish (GloFish), growth, meat color, fertility control, feeds for aquaculture
    • "Eco" toxicogenomics: Biomonitor fish (inducible and reporter-based monitoring): Environmental pollution and bioterrorism monitoring

Mus musculus – the House Mouse

  • Of the models so far clearly the largest similarity to humans.
  • Common ancestor existed around 80 million years ago.
  • Similar genome structure and organisation and similar number of genes.
  • Amenable to genetic manipulation.

Large Animal Models of Human Disease

  • Despite the use of rodent models in biomedical research there has recently been a surge in the use of large animals to model human disease.
  • This is linked to gene editing tools such as CRISPR/Cas9 and similar methodologies in large livestock animals to produce newer models that may replicate the human condition with more fidelity.
  • For example domestic pigs and mini-pigs bear remarkable similarity to humans.

Other Models with Specialised Uses

  • Rats
  • Rabbits
  • Chickens
  • Sheep
  • Dogs
  • Non-human Primates

The Many Model Systems of COVID-19

  • Scientists have been exploring various models to study COVID-19.
  • See: https://www.the-scientist.com/news-opinion/the-many-model-systems-of-covid-19-68125

3Rs – Replacement, Reduction, Refinement

  • The principles of the 3Rs (Replacement, Reduction, and Refinement) were developed over 50 years ago as a framework for humane animal research.
  • They have become embedded in national and international legislation regulating the use of animals in scientific procedures.
  • Support for animal research in the UK is conditional on the implementation of the 3Rs.
  • See: www.nc3rs.org.uk/the-3rs

Definitions of the 3Rs:

  • Replacement: Methods which avoid or replace the use of animals
  • Reduction: Methods which minimise the number of animals used per experiment
  • Refinement: Methods which minimise suffering and improve animal welfare

Genetic Manipulation of Model Organisms (Part 2 Preview)

  • How to genetically manipulate model organisms
  • Making transgenic organisms
  • Knock-in and Knock-out mutations
  • The use of RNAi for performing Knock-down experiments
  • The use of CRISPR-Cas technologies for genome editing