Professional Documents
Culture Documents
Animal Experiments in Medical Microbiology
Animal Experiments in Medical Microbiology
Animal Experiments in Medical Microbiology
Medical Microbiology
Introduction
Laboratory animals long been used for various
experiments in medical microbiology
(Declaration of Helsinki)
The 3 Rs –The guiding principles in
animal research today:
1. REDUCE the number of animals used to a
minimum
2. REFINE the way experiments are carried
out
3. REPLACE animal experiments with non-
animal techniques where possible
Animals Used in Medical
Microbiology
Rodents and Lagomorphs
Mouse
Rat
Voles
Gerbils
Guinea pigs
Hamsters
Rabbits
Ungulates-Sheep,goat,horses,pig
Carnivores-Cat,dog
Primates
-Monkeys,Chimpanzees
Birds
-Fowl
Amphibia-Frogs,toads
Other mammals-E.g. Armadillo
Mice in Medical Microbiology
Mouse(pl-Mice) – Mus musculus
“Mouse” from Sanskrit “Mush”
Most commonly used lab animal
Mammal with very similar genetic
makeup to humans
Manifold genetic variations
Convenient size
High fertility rate
Rapid generation time(9 wks)
Cheap and easily available
History of mice in research
1800s-Mice fanciers
1900-Retired school teacher Abbie Lathrop housed over
11,000 mice for their unusual appearance
1902-Lucien Cuenot,in France first to demonstrate
Mendelian ratios for inheritance of coat colour
characteristics in mice
1921-Clarence Cook Little-credited with conceiving of
and creating the first inbred strains of lab mice(DBA-
Dilute Brown Agouti)..credited with establishing Jackson
laboratory
1977-First mouse gene isolated
2002-First draft of mouse genome published
Of Mice and Men
Mouse genome project completed in 2002(strain
used C57BL)
Handling
Identification
Marking with stains(Dye marking scheme by Lumsden
1973)
Holes in ear with ear punch
Anaesthesia
Short acting –Ether
Long acting-Phenobarbitone i.p
Ketamine i.m
Atropine to reduce secretions s.c or i.m
Administration of materials
Body fluids
Blood
Urine
CSF
Serous fluid
Cultures
Fluid cultures
Growth on solid media
Tissues
Brain,liver,spleen,liver-Glass tissue grinder
Muscle,lung,skin,lymph nodes-electrically powered blender
Routes of inoculation
Subcutaneous-0.2 ml
IV-0.7ml(max)
Intraperitoneal-2ml
Prior withdrawal of food overnight with access to water to
decrease chances of injection into viscera
Intracerebral-0.03ml
Intranasal-0.1ml
Injection of infant mice
10X 0.5 mm needle used
Subcutaneous-0.03ml
Intraperitoneal-0.05ml
Intracerebral-0.03 ml
Collection of blood
From tail-0.3 ml
Retroorbital plexus-0.7 ml
Heart-upto 1.5 ml
Euthanasia
Cervical dislocation
CO2/Coal gas cabinet
Chamber containing cotton wool saturated
with Chloroform
CHOICE OF MICE
GENETIC STATUS
Inbred mice
Outbred mice
Mutant mice
Nude mice
SCID mice
Hybrid mice
Knockout mice
Transgenic mice
MICROBIOLOGICAL STATUS
Conventional
Specific Pathogen Free(SPF)
Gnotobiotic(Germ Free)
Inbred Mice
Produced by Brother X Sister matings for atleast
20 generations in such a way that
all individuals trace back to a single common
ancestor
Inbreeding –F(Coefficient of Inbreeding)
Inbred strains –Isogenic(genetically identical)
Advantages
Genetic stability
Uniformity
Characteristics of inbred mice
Isogenicity-all mice of a particular inbred
strain-identical or isogenic at 99% genetic loci
Homozygosity
Stability(genetic)
Uniformity-only few inbred animals required
for statistical calculations compared to
outbred
Sensitivity-to experimental t/t and change in
environmental conditions
Uses
Used in cancer research for over 60 years
Hybridoma technology
Tissue transplantation studies(transfer of
tumours b/w syngeneic and xenogeneic
animals)
Specific strains developed for
High incidence of tumours
Unique pattern of behaviour
Specific immune responses
Susceptibility to particular diseases
List of Inbred strains
Albino White BALB/c
Agouti black CBA
Agouti C3H
Dilute Brown DBA/1
Albino white NZW
Wistar Furth,Wistar Kyoto
X linked immunedeficiency(Xid)-Chr 10
SCID (scid)-Chr 16
Mutant mice can be inbred or outbred
Types of Mutant inbred mice
Isogenic-2 inbred strains genetically identical
Coisogenic-single locus change due to point
mutation
Congenic-strains differing from normal
counterpart by small fragment of single
chromosome
Nude Mice(Athymic Mice)
Well studied model of primary immunodeficiency
Genetic mutation that causes deteriorated or absent
thymus →inhibited immune system
-greatly ↓ no. of T cells
Genetic trait designated nu controlled by a recessive
gene(FOXN1 gene) on chromosome 11
Homozygous mice(nu/nu)→
hairless ;vestigeal thymus
Animals maintained under
conditions protecting them from
infections
Nude mice-lack CMI responses
Unable to make abs to most antigens
Tolerate both allografts and xenografts
USES
Hybridomas or solid tumour from any origin grown as
ascites or implanted tumor in nude mouse
Animal model in study of autoimmune diseases
Demonstrate helper T cell function
Demonstrate func of T lymphocytes in transplant
rejection
Cultivation of lepra bacilli and to detect its drug
sensitivity
SCID Mouse
Model for primary immunodeficiency
SCID mouse-Early B and T lineage cells present
Virtual absence of lymphoid cells in
thymus,spleen,lymph nodes and gut tissue
Neither make abs nor carry out DTH or graft
rejection
Useful in studies of cellular immunology
Window into possible causes of combined T and B
cell immunodeficiency
1980s-SCID mouse →
In vivo model of the human
immune system
Human fetal thymus and
Ig of human origin
SCID mouse reconstituted with human lymphoid tissue
2 major methods
Pronuclear microinjection-foreign DNA
introduced directly into mouse egg just after
fertilisation
Introduction of DNA into embryonic stem cells
Knockout Mice
Technique in which a desired gene targeted to specific
sites within the germ line of a mouse
First knockout mouse-Mario Capecchi,Martin Evans and
Olivier Smithies in 1989
Primary use-replace a normal gene with a mutant allele
or disrupted form of the gene,knocking out the gene’s
function
Infering the probable function of a gene
Mouse models named after the gene which is targeted
e.g.-p53 knockout mouse-named after p53 gene
simulates Li-Fraumeni syndrome seen in humans
Gene targeted knockout mice
Isolation,culture of ES cells from inner cellmass of mouse blastocyst
↓
Introduction of mutant or disrupted gene into ES cells
↓
Selection of homologous recombinant cells in which gene of interest
knocked out
↓
Inj of homologous recombinant ES cells into recipient mouse
blastocyst
↓
Surgical implantation of blastocyst into pseudopregnant mouse
↓
Mating of chimeric offsprings heterozygous for the disrupted gene
produce homozygous knockout mice
Hybridoma Technology
Large scale production of monoclonal antibodies against
any desired antigen
Rabies vaccine-
CPCSEA guidelines