Genetic manipulation - Physiology Flashcards
Dutch APP mutation
Over-expressed APP (mice)
Cleaved - alpha-secretase site
Still develop cognitive effects similar to human AD
Swedish APP mutation
Over-expressed APP (mice)
Cleaved - beta-secretase site
More likely to get A-beta accumulation (plaques)
V717F mutation
Over-expressed APP (mice)
Cleaved - gamma-secretase site
Transgenic mice over-expressing APP
Potential therapy?
Extra genetic material
Therapy - active immunisation of A-beta-42 peptide
Susceptible to AB42 (Swedish mutation)
Inoculate against disease - antibody against AB42
Some success in mice models
BUT - are APP over-expressing mice a good model of human AD disease?
Better model = Double transgenic mice = human APP + mutant tau
Gene targeting process
Replacing an endogenous gene
Uses regions of homology (flanking sequences) for homologous recombination
Add thymidine kinase (tK) outside regions of homology
Targeted insertion = will not integrate
Random insertion = will integrate
Add antibiotic - neomycin AB resistance for all transfected cells (non-transfected cells die)
Expose to gancydovir - non-toxic alone, toxic with tK - kill cells non-homologously recombined (random insertion)
Problems with knock-out mice
Functional redundancy
APP-KO = APP-like-protein-1/2, in absence of protein take over function
Embryonic lethal
Presenilin-1 (implicated in dementias) - gene also has a role in development, KO impairs normal development, embryo dies in utero
Cell specific KO
CreRecombinase-LoxP system
Insert Cre-recombinase downstream of cell-specific promotor
Gene targeting - insert LoxP flanking sequences around gene of interest
Cross mice
Cell-Specific KO
ie. LTP
CA1 place cells, hippocampus - important in spatial memory
NMDA receptor = NR1
Aim for CA1-specific NR1 KO
Promotor for cell of interest (CA1 place cells) - place Cre recombinase downstream
LoxP flanking sites via gene targeting either side of NR1 gene
Cross breed mice
Produce mice CA1-specific-NR1-KO
Measure effectiveness using Morris Water maze - measures spatial learning + memory
Results:
CA1-NR1-KO
Severe spatial learning defects
Reduced spatial selectivity/coordinated activity of place cells
Can visualise using electrophysiology recordings - see reduced firing
CA3-NR1-KO
Impaired pattern completion
Place cells less accurate in novel contexts
Why use mice?
Breed quickly
3 week gestation period
Ovarian cycle - 4 days
Pups take 3 weeks to ween - rapid turn around
BUT
Value of disease models in mice depends on similarity to humans (face validity)
Promotor for eukaryotic cells
Pcmv
Antibiotic resistance gene - bacterial cells
Ampicillin
Antibiotic resistance gene - eukaryotic cells
Neomycin
