Priciples of animal experiments

Question 1

What’s the need for animal experiments?

Answer 1

• 1 billion illnesses p.y.
• 10 million death p.y.
• can‘t infect human
• impossible to analyse exclusively in vitro

Question 2

legal issues

Answer 2

• application needed
• newly performed experiment & why needed

Question 3

SPF (specific-pathogen-free) breeding & keeping

Answer 3

• free from pathogens
• hygiene and health monitoring
• airtight cages, filtered air, pos pressure
• import of animals via embryotransfer

—> mice often in individually-ventilated cages (IVCs)

Question 4

Cultivation of pathogens

Answer 4

• in vitro acellular (bacteria)
• in vitro in host cells (viruses, intracellular parasites)
• in vivo in animals

Question 5

Infection of animals (routes)

Answer 5

• close to nature
• intravenous (i.v.)
• subcutaneous (s.b.)
• orally (p.o.)
• intraperitoneal (i.p.)
• aerogenic

Question 6

Analysis of infection

Answer 6

pathogen loads:
- lung, skin, blood, intestine/feces

immune response:
- non-lymphoid infected tissue (as above)
- blood, serum
- lymphoid tissue

pathology:
- infected tissue
- blood, serum
-liver

Question 7

Analysis of susceptibility

Answer 7

• haemagglutination, plague-assay (virus)
• colony forming units (bacteria)
• parasitemia (blood parasites)
• limiting dilution of single cellsuspension (intracellular parasites)
• worms in organs/eggs in feces (worms)

Question 8

Analysis of immune response

Answer 8

humoral factors:
- cytokine/chemokine expression (RT-PCR of organ-homogenates, ELISA of sera)
- production of ABs (ELISA of sera)

cell-mediated immune response:
- recruitment of immune cells (FACS, Immunohistochemistry)
- activation of immune cells (FACS, Immunohistochemistry, restimulation of T cells -> ELISpot, Cytotoxicity)

Question 9

Pathological analysis

Answer 9

histological examination:
- HE-stain
- azan stain to detect fibrosis

blood parameters:
- liver-derived enzymes —> liver necrosis
- potassium —> kidney damage
- glucose —> liver damage

Question 10

mouse strains

Answer 10

inbred:
BALB/c

C57BL/6 —> IL4 -/-

wildtype
genetically manipulated

Question 11

Ho do you generate knock out mice?

Answer 11

• homologous recombination
• microinjection of ES into blastocysts
• gene editing (CRISPR/Cas9)

Question 12

How does homologous recombination work

Answer 12

1. insert gene into exon -> exon gives recombination sites
2. target vector needs suicide cascade
   3a. homologous recombination inserts vector at target gene site without suicide cascade
   3b. random recombination inserts vector random with suicide cascade
   3c. no insertion

selection of cells with site specific insertion
efficacy: 1%

Question 13

How does microinjection work?

Answer 13

1. ocyst of (black) mice
2. insertion of (brown) ES cells into ocyst
3. manipulated embryo growth in foster mother
4. result is a mosaic/chimeric offspring (heterozygous)
5. chimeric male mouse + black female mouse to get homozygous offsprings (black or brown)

Question 14

How does CRISPR/Cas9 work?

Answer 14

1. CRISPR induces double strand breaks
   2a. non-homologous end joint leads to disrupted DNA with deletion or addition
   2b. homologous direct repair with donor DNA leads to repaired DNA with target gene

• zygote is manipulated -> pure manipulation
• only one generation of mice needed