L27 - Genetically modified animals Flashcards
(17 cards)
what can be used to create ‘knockout’ mice/animals
CRISPR
1 pro and con of using mouse as model
pro:
mice have a genome 90% similar to humans
= can develop human diseases and be used for reference
cons:
although simiklar anatomy and genetics are different
= somthing that works in ice may not work in humans
how do NHEJ and HR work to introduce knockouts in mice/animals
HR:
repair template flanked by honmologous seuqnces added
= desired gene is incorparated
NHEJ:
ends are proscessed and INDELS are introduced
= small disruption in sequence can knout gene
what is the hayflick limit
cells in culture cannot replivate indefinietly due to telomeres at end
describe how to create genetically modified Embryoinc stem cells
- isolate ESCs from the inner cell mass of a blastocyst
- create a plasmid with homology arms complimentary to target
= plasmid contains Neomycin gene within homogy arms AND TK gene OUTSIDE homology arms
- introduce bacterial plasmid into ESCs by elctroportation
- add antibiotic for selection to see if cells have plasmid AND if they had succesfull insertion/homologous recombination
- surving colonies are allowed to grow and PCR is used to validate if gene is present in genome of mouse cells
describe the positive/negative selction in embryonic stem cells for mice with Neomycin and TK
3 possible results in the cells:
- NK inserted in correct spot and cell survives due to NK breaking dfown antibiotic
- NK is inserted into correct spot WITH TK = whole plasmid was isnerted
= TK creates a toxin = these cells die
- no insertion of plasmid = no antibiotic resisatnce = cells die
–> the only cells that surive after selection are those with the NK gene that was inside the homogy arms AND NOT anything oustide
how do we produce homozygous mice after obtaining Embryonic stem cells with knocked out genes
- obtain another blastocyst from another mouse and miucroinject our edited ESC into surroagte fenake
= chimeric mouse produced (mix of normal and knocked out cells)
- breed chimeric mouse with wild-type mouse
= select pups from this based on fur colour to identify which mice have the ESCs from chimeric mice
- cross 2 heterozygote knockout mice together
- screen by PCR to ensure that mice is knocked out of the target gene
how do we create knockouut mice in CRISPR era
use CRISPR to create ds breaks in target gene
= NHEJ creates INDELs
if we want to introduce a gene we can also add in a repair template plasmid with homogy arms or a oglioncueltide with a point muation to be HR template
how does HR work to introfduce a point mutation with oglionculeotide
- ds occors and ends are chewed back to create 3’ OH overhangs
- strand 1 binds to the homologous oglionucletide and uses it as template = DNA pol extends strand 1
- strand 2 anneals to the extended new strand via comp base pairing
= polymerase fills in any gaps
what is gene replacement in genetically modified humans
delivery of functional gene to replace non-working gene
define gene silencing and gene addition in terms of gentically modified humans
silencing:
inactivation of mutated gene that has becomne toxic to cells
addition:
adding gene to be expressed to impact cellular function
describe
the idea of using CRISPR to solve sickle cell disease
sickle cell disease occors due to problem with adult haemoglobin
= we can re-introduve fetal haemoglobin and releive the symptoms by turning off the gene that stops fetal haem production
describe method of using CRISPR to infuce fretal haemoglobin to help sickle cell disease patients
- in sickle cell adult haem have a harmful mutation in beta-globin gene –> become HbS instead of HbA
= in low oxygen envirnments haem molecules stick together to form long rigid fibers inside red blood cells
- BCL11A gene turns off fetal haem
- introduce CRISPR to cut or disrupt this gene in bone marrow stem cells = fetal Haem produced again
what type of editing strategy is CRISPR on bone marrow cells to releive sickle cell patients
ex vivo
= bone marrow cells are isolated and taken OUT of body
edited and re-introduced
how can we undergo ‘in vivo’ editing therapies on humans
nucleases delivered by biral or non-viral approaches directly into patient
= for whole body or targetted effect
what are the limitations of using viral and non-viral vectors for ‘in vivo’ editing strategies
viral:
can cause immune response
high cost
= viruses infect cells with gene editing CRISPR-cas system for editing
non-viral:
less expesnive
less chance if immune response
= lipid nanopartciles carrying CRISPR-cas system into cell by fusing with membrane
