Mouse Design Flashcards
genetic homology between humans and mice
99%
physiological similarities between humans and mice
cerebellum, cortex, thalamus, medulla, pons and olfactory bulb
what vectors are used to transfer genes in transgenic modification?
bacterial plasmid
where is the gene of the interest transferred during random integration?
male pronuclei of fertilised egg
once the DNA is randomly inserted into genome of pronuclei, where are the eggs implanted?
pseudo-pregnant females
why do founder lines differ despite being infected with same DNA?
DNA nserted at different integration sites
components of transgene:
gene of interest, reporter construct, control element
how are transgenic lines produced?
founder offspring backcrossed to WT
how are offspring screened for transgene?
PCR, southern blot and probe
random integration refers to:
each egg possessing different copies at different locations
transgene
cDNA or cloned genomic DNA
what determines where and when the transgene is expressed?
promoter
CMV
strong promoter leading to constitutive expression in mammals
difference between CMV and cell-specific promoter
cell-specific promoter allows for regulated gene expression
cell-specific promoter
GFAP
why is DNA microinjected into the pronuclei of fertilised eggs?
so genes introduced into germ line
ALS mouse model
G85R placed into SOD1 and injected into male zygote pronuclei
ALS phenotype (4 signs)
poor righting and hindpaw grasping
paralysis 2 weeks after clinical onset
aberrant hindlimb posture within 3 days of clinical onset
increased endogenous SOD1 expression correlated with
younger onset
how do interference RNA knockdown genes
microRNA or shRNA disrupt transcription
dominant negative construct
mutant protein impairs function of wild-type protein
cre recombinanse
recombination at loxp sites
negatives of transgenic over-expression
ectopic expression
multiple copies inserted
insertional mutagenesis
how can random integration lead to insertional mutagenesis?
if inserted into exon or essential element
what kind of insertion does not affect gene expression?
intergenetic
How does transgenic insertion into exons or introns cause confounding phenotypes?
insertional mutagenesis
What kind of confounding phenotypes does random integration usually cause?
KO
Zfp36 KO models:
arthritis, dermatitis
Zfp36 KO models:
arthritis, dermatitis
ROSA26
produces mRNA which does not code for a protein
KO generation
homologous recombination to produce the target gene in ES
pos and negative selection of ES stem cell
implantation into blastocyst and injection into foster mother
how are transgenes incorporated into ES?
electroporation
neomycin gene
positive selection (will survive in presence of geneticin)
what does survival from geneticin not control against?
random integration
how does gancyclovir select against random integration?
converts tK into toxic compound
presence of tk means homologous recombination has not occurred as tk is outside of the arms
why do chimeric mice have fur of both colours?
developed from injected and native ES stem cells
chimeric mice bred with WT to produce
F2 KO
constitutive KO + KI disadvantages
may cause embryonic/perinatal lethality/compensatory upreguation of related genes, Neo gene can alter expression levels (in KI)
solution of embryonic/perinatal lethality
cell-type specific expression and inducible knockout
solution to compensatory changes
inducible knockout in adulthood
how to prevent neo-induced changes to gene expression?
neo removal
