Advanced animals models Flashcards
Why do we use transgenic animals?
To study the function of a gene, or to better understand mechanisms such as modelling human disease or testing therapeutic approach.
What are adult stem cells and IPS?
Adult stem cells are undifferentiated cells that are multipotent (limited in ability to differentiate).
IPS cells are differentiated cells that are de-differentiated and can be programmed to differentiate to a specific type of cells
What medium is used to maintain stem cells?
LIF-leukemia inhibitory factor.
How are transgenic knock out mice created? What does the vector contain?
They put a marker where the gene is on a vector and inject in the embryonic stem cells, only the cells with marker will survive this happens through recombination
The vector has homologous arms and a selector marker
What are the problems with homologous recombination? What’s a possible solution?
Many non homologous recombinations form.
Replacement vector
What is the replacement vector made up of?
Replacement vector has two selective markers: neomycin and HSV-TK and two segment of the gene
How does replacement vector work?
So recombination occurs it can be homologous or non homologous. The cells are plated on gancyclovir, When homologous it will be neomycin resistant but doesn’t have TK. However when random non homologous recombination occur then TK will be there and it will be converted to a toxin by gancylclovir and those cells will die.
How to transfer ES into mice through germline?
Inject the ES cell with the knockout gene in surrogate mother and mate with wildtype mouse
There will be chimeric mice: positive and negative. Mate heterozygous with heterozygous mutant
will have 3:1(mutant)
Problems with interpeting knockout experiments?
It’s difficult to associate the function to the gene, could be indirectly caused by it, no phenotype doesn’t mean gene isn’t effected. Knockout can kill embryo.
What can be used as an add on experiment or instead of it?
Chemical induced mutant animals
What does the vector have to constitute of for knock-in experiments?
Gene of interest, marker and promoter
What are the ways that knock ins can be done?
Through embryonic stem cell transformation or through inject the egg
How do both of the knock in experiment approach differ from each other?
Injection of transgene happens in the pronuclei of the sperm in a fertilized egg and then all offsprings will express the gene everywhere however it will happen through non homologous recombination. But the ES transformation through neomycin selection and then inject these cells into the blastula gene will be inserted through homologous recombination; chimeric
What are the positive and negatives of injection and transformation?
Injection: \+: No chimeric -: Less reliable integration or viability of eggs ES transformation: \+: More control over the integration -: It only works on mice
How does inducible knockouts work?
Flox(inverted sequence repeat) flank the gene of interest, when CRE recombinase is expressed it will cleave both flox and the gene will be removed
