Transgenic and Knockout Mice Flashcards
(48 cards)
what is an ‘IN VITRO’ study ?
-literally mean ‘within the glass’
performed not in a living organism but in a controlled environment such as a test tube or petri dish
what is IN VIVO research ?
-literally mean ‘within the living’
experimentation using a whole organism that is living
it is better suited for observing the overall effects of an experiment on a living subject
what are 2 forms of in vivo studies ?
animal experimentations and clinical trials
why are mice the most commonly used mammalian species used for animal experimentation ?
they replaced guinea pigs because…
- smaller
- cheaper
- faster breeding
how many more nucleotides does the human genome have compared to the mouse genome ?
400 million - we are much bigger and more complex
when did humans and mice genetically diverge ?
about 75 million years ago
- we had a common ancestor
how much of the human genome is similar to the mouse genome and what does it mean ?
99% of human genes have a mouse counterpart
only 300 genes unique to either organism
what does “KNOCK-OUT” mean ?
it means a gene can be deleted in mice to determine its function- one of the simplest ways to do this
why is the mouse a good genetic model ?
STRAIN- can inbreed mice which is useful because the mice are identical and therefore you minimise other effects when looking at a specific gene
GENOME- similar counterparts to humans
LIFE CYCLE- only have a 20 day gestation period and produce 4-8pups but sometimes as many as 15. also onnly have a life span of about 2 years so aging disease can be studied and females are sexually mature by 7-8 weeks
ASSISTED REPRODUCTION- ICIS- select sperm and inject directly into an egg
GENETIC MANIPULATION- interfere with genome but knockout genes, putting in mutations etc
RESOURCES- lots of info on them such as genome is fully sequenced
what was piebaldism caused by and what does it do ?
caused by a mutation of the KIT gene
lack of pigementation on the forehead and stomach
present in both humans and mice
what happens with a congenital leptin deficiency ?
mutation in the leptin gene causes obesity
it is a recessive mutation so to have the disease you need 2 recessive alleles
it causes clinically obesity because leptin is an appetite suppressing hormone so therefore it causes them to eat more because they lack this hormone
the same phenotype was shown in mice
what are some similarities and differences between mouse and human brain ?
mouse brain is smaller than the human brain
everything in the human brain is also present in the mouse
humans have a smaller olfactory bulb because it is not as important to humans
human cortex is folded unlike the mouse and this is because it increases the surface area which is necessary because the human brain cant be too big because then childbirth would be impossible
what does the term transgenic mice mean ?
refers to mice that are genetically manipulated by technology which allows integration and expression of exogenous DNA fragments into their entire genome
- DNA that wouldnt naturally be present and it is passed onto subsequent generations
what do transgenic mice allow?
enable us to study gene function or regulation and to model human diseases
may confer a gain of function (may be due to overexpression) or loss of function (integrated DNA interrupts another gene )
what manipulations of a mouse can be done ?
transgenics= gene over expression
knockouts= deletion of a gene
knock ins= modifications of a gene - not the same as a new gene being added, it is the addition of a mutation to an existing gene
inducibles= induce expression or knock out a gene using a drug
how to genetically modify a mouse?
random integration
homologous recombination
in combination
what does random integration entail ?
injection of DNA into a fertilised mouse egg so the DNA randomly integrates into the genome
- over expressiion
- region specific promoters= splice gene with specific promoter so gene is only expressed in some cell types
- inducible system
what does homologous recombination entail ?
use of embryonic stem cells- target a particular sequence to knockout gene or knock in mutation
c
-consitiutive knockout
-knockins
what does in combination entail ?
make a mouse express the enzyme cre by random integration and then using homologous recombination you produce a mouse with lox P sites which surround the gene of interest that we want to knock out and then these 2 mice are crossed together
you get mice which express cre in particular cell types
cre combines with lox P sites and knocks gene out
explain the process of producing a transgenic mouse using random integration ?
1- transgene spliced into a plasmid
2- transgene construct is injected into the pronucleus of a fertilised egg
3- eggs are implanted into a pseudo-pregnant mouse
4- offspring are screened for the presence of the transgene
DNA randomly integrates into the genome so you produce founders that have the DNA integrated in different area
what can transgenes carry ?
gene of interest
reporter construct such as GFP which can be spliced with the gene of interest so you can determine where the gene of interest is expressed
control element- cre recombinase
what founders are crossed?
the heterozygous ones to try and get a homozygous
what is a pseudo-pregnant mouse ?
female mated with a vasectomised male so her physiology makes her act like she is pregnant so her uterus is good to carry offspring
how is a transgenic construct designed?
regulatory promoter sequence- controls when and where the gene will be expressed
cDNA of the gene- no introns, all the exons are joined together
