Genetics Flashcards
Outbred animals (mice/rats) - advantages and disadvantages
Low price
High variation robust animals
High heterogeneity (if truly outbred)
Low study reproducibility (the animals are not alike)
Bred by rotational breeding (small colonies)
Inbred animals (mice/rats) differents from outbred
High price (compared to outbred)
Low variation - increased risk of resulting in lethal recessive gene combination
Relatively homozygous (97% of homozygoticy)
High study reproducibility (the animals are very similar)
Brother-sister or father-daughter breeding for at least 20 generations (trafic light system pyramid)
Breeding program for laboratory animals INBREED (trafic lights)
Close relatives (brother x sister or father x daughter) are bred for at least 20 generations. The homozygosity can reach approx. 97% - highest achievable for the animals to survive
- Stem pair of brother and sister
- The nucleus is the pups from stem pair. The pups are mated brother x sister.
The pups from nucleus are the basis of the strain and can be bred randomally – as it is ok to use cousins.
PC1: The pups from the green “production” colony can be used as breeders in the yellow production colony and for research
PC2: The pups from the yellow production colony can be used as breeders in the red production colony and research
PC3: The pups from the red production colony can only be used for research.
Breeding program for laboratory animals OUTBREED - rotation and hybrid
Heterozygotes with a relatively large gene-pool. Often quite robust animals.
Hybrid – F1:
Crossing two different inbreed strains with each other. This results in robust animals with a high degree of heterozygosity.
Small colonies: Rotational breeding
In rotational breeding the colony is subdivided into say four subcolonies, A, B, C and D.
Females produced in A, can be used for breeding in B, from which females are used for breeding in C, from which females are used for breeding in D.
Males are exchanged between A and C and B and D.
Of course, offspring from all four subcolonies can be used for research studies
Genetically altered animals - how to make them!
Simple Pronucleus microinjection
Simple Pronucleus microinjection:
1. A female is euthanized one day post mating - eggs flushed from salpinx - injection of the desired gene into the egg
2. A new female is mated with a sterile male and the manipulated eggs are placed within her salpinx
The gene is RANDOMALLY placed within the genome
you need to breed for generations to get homozygotic animals.
Used for randomly insertion of a functional gene
All species
INSERTION ONLY
Embryonic stem cell transfection
Genetic alteration of the animal (mice, rats and humans)
- Embryonic stem cells are harvested from blastocytes
- Cells are grown in culture
- Gene modification: knock-in/Knock out
- The cells with mutations are harvested and injected into another blastocyte and inserted into a pseudopregnant female
5 Chimaeras are born = mice with WT and gene modified cells
- often differented by fur color (embryonic cells from agouti and blastocyte used for insertion from B6) - Breeding for a homozytogotic offspring requires multiple generations
- Male: WT/GM mated with a WT female –> all WT/WT pups are discharged.
- breeding WT/GM mice hopefully results in GM/GM
Targeted mutation and Knockout (in mice and rats only)
What is a mutant?
example (a selective inbreed NMRInu)
A spontaneous mutation with an alteration in the genes (recessive gene = only in homozygotic animals):
Naked mouse with not thymus = no t-helper cells or FUR.
Selective inbreed - rats and mice with a spontaneous phenotype
Specifically bred for the phenotype.
Wister rats: spontaneously development of hypertension - BP >180 mmHg compared to the normal 110mmHg
NOD mouse (non-obese diabetic) Diabetic mouse: strains develops diabetic 1 by a autoimmune destruction of the beta-cells within the pancreas (as seen in humans)
Transgenic mice:
- knock in
- Knock out
Alteration of the genome inserting a gene from another species, it can be both in the coding and the non-coding region within the DNA
Knock in: a functional gene has been inserted - often a humane gene instead of the mouse gene. ONLY homozygotes
Knock-out: a gene is made non-functional. ONLY homozygotes
Nomenclature: general
First: strain of mouse (if a hybrid = F1)
Origin (breeder code)
Divider between strain name and breeder coder: “:” (colon) outbred and “/” (slash) inbred
Tm: targeted mutation
Tg: Transgenic
Gene: in capitals = humane, small letters = mouse gene
Knock out: supscript (hævet) desciption of method, gene and originating laboratory
Knock-in: Gene name in italics (kursiv)
C57BL/6-TgN(APOA1)1Rub/J
B6 mouse (INBRED)
Tg: tageted mutation
N: non-homololous method (could be microinjection = randomally)
Humane gene: APOA1
Rub: Founder breeder
J: Breeder code
B6.129P2-ApoA1 tm1Unc/J (method and founder is subscripted and in italics)
B6 and 129 hybrid
mouse gene: ApoA1
tm = Targeted mutation (often CRISPER-Cas9)
Method and founder is in italics = KNOCKOUT
Breeder code
Female mouse.
age fertile?
how long is estrus?
how long pregnant?
when to wean?
Fertile approx. 7 weeks
Eatrous for 12 hours (allows mating)
Vaginal plug
pregnant for 3 weeks
Wean pups when 3 weeks
Hybrids how to make them
mate two inbred strains = robust animals with a high degree of heterogozity
DNA:
95% non-coding regions
5% coding
where do you alter the genes? transgenic (including knock-out) and knock-in
Transgenic: In the non-coding regions which will effect turning on/out of the genes
Knock-in: in the coding region = functional gene
Cloning and nuclear transfer (pig)
- Cells are harvested from a fibroblast and cultivated
- Gene modification of the cell sulture
- Saw egg: remove the nucleus and insert a gene modified cell nucleus instead.
- The fertilized egg is developed into a blastocyte
- the gene modified embryo is inserted into the uterus of a pseudopregnant saw
6 gene modified piglet is born
HUmans and large animals - not used very much anymor
Nuclease technics for GMO (Gene modification)
- an enzyme which can induce breaks in the DNA string (originates from bacteria)
CRISPR-Cas9 - induces a double break in the DNA string by the guidance of a short RNA string which grabs on to the DNA string and the CRISPR-CAS9 begins to cut it.
The cut of the string can be very specific = Targeted mutation
KNOCK_IN: A specific gene can now be inserted
Can be used in relation to both microinjection and embryonic stem cell in culture.
ALL SPECIES!
Most used inbred mouse in the world?
B6
SUM UP GMO methods
Micro injection: random placing of gene. all species.
- not useful for knock-in or knock-out.
Embryonic stemcell tranfection: Targeted alteration, insertion of a functional gene possible, both knock-in and knock-out is possible. ONLY mice, rats and in principle humans
Colning and nuclear transfer : targeted alteration, insertion of a functional gene possible, both knock-in and knock-out is possible. all species!
CRISPR-Cas9 (easy and cheap): targeted alteration, insertion of a functional gene possible, both knock-in and knock-out is possible. all species!
Principles of genetic monitoring
Sample types
Principles of genetic monitoring: testing methods for a specific genotype
PCR:
- single gene (transgene mouse)
- easy and cheap
- protocol is often available
- relatively simple method
Southern blot:
- single gene
- easy and cheap
- protocol is often available
- many steps = old method, use PCR
testing method characterization of strains or stocks
Southern blot:
- single gene
- easy and cheap
- protocol is often available
- many steps = old method
SNIPS: single nucleotide polymorphism
- testing 1000 of polymorphisms on all chromosomes
- costly
The SNP are stable over generations. - the snips are often in the coding genes
Short tandem repeats (STR) chips:
- the tandem repeats are often in the non-coding regions –> unkown impact on gene expression.
- costly
- may variate between individuals even in inbred mice
