genetic engineering Flashcards
(21 cards)
what does cre do
catalyze recombination between 2 loxp sites
what is cre
dna recombinase (enzyme)
three types of genetically engineered mice
knockout
knock in
conditional knockout
why do they make gen ed mice
to study gene function and model human diseases bc their genomes are similar to ours
neo r
neomycin resistant gene
postitive selection
kills the cells without the neor bc neor is positive
negative selection
kills the gene with the tk bc its negative and keeps the healthy cells
tk
thymidne kinase found in herpes - suicide cell
knockout mice steps
-add neor and tk into target gene to make targeting vector sequence
-add the targeting vector into es cells via electroporation
-only cells that have successfully incorporated survive when neo and ganciclovir is added
-cells containing the targeting vector are added to embryo
-results in a chimeric mouse (spotty)
electroporation
creates electricity/pores for dna to get in - like distracting and slipping in through memebrane
es cells
embryonic stem cells (come from embryos)
conditional knockout
deletion of gene in selected cell types
crispr
clustered regularly interspaced short palindromic repeats - part of the bacterial immune system
cas9
enzyme- a becterial endonuclease that cuts dna at a specific site within a larger target sites
nuclease
an enzyme that cleaves (cuts) the bonds of the backbone of nucleic acids
pam
downstream of the protospacer sequence based on where the pam is depends on where the cleavage will be
guiding region
20 nucleotides long and is complementary to the target dna sequence that defines where cas9 cuts
scaffold region
a region that forms a hairpin loop that binds in a crevice of cas9
target dna
the sequence of dna that will be cut by cas9
sgRNA
includes guiding region and the scaffolding region
floxed
cre will look for the loxp and pull out the stuff in between it
