genetic manipulation pt 2 Flashcards
how does cre-recombinase work
cre is a sight specific recombinase enzyme that recognises a 34bp DNA sequence loxP
if gene in-between 2 loxP sites, cre will snip out the gene
Flp recombinase
works same as Cre-recombinase
binds to FRT sites (FLP recombinase targets)
yeast system as opposed to bacterial
principles of Cre/Lox conditional knockout
need two strains of mice
1. mouse with gene of interest floxed
2. mouse expressing cre-recombinase from tissue specific promoter
breed stains together –> mice where gene is knocked out in target tissue
Cre/Lox knockouts: how is floxed mouse created
done using homologous recombination in ES cells
DNA targeting vector with gene flanked by loxP sites. Neomycin resistance gene too for identification. NRG will need removed later, so flank this with Flp-recombinase sites (FRT)
electroporate target vector into ES cells and apply neomycin
inject surviving cells into blastocyst –> chimeras –> breed to get heterozygous floxed mice
breed with mouse line expressing Flp recombinase from a constitutive promotor to get rid of NRG
–> floxed baby mice w/o neomycin resistance gene
internal ribosome entry site
allows two proteins from 1 ribosome
ribosomes jump 2 sites and make cre, but also GFP in same cell –> cell expressing cre is also bright green
making cre transgene by injection of DNA
make cre construct: processing signals, promoter, Cre, GFP, IRES, polyadenylation signal, intron
difficulty is getting a good promotor only expressed in tissue of interest
a solution is to knock in cre gene in replacement of a gene know is expressed in tissue of interest
knocking in the cre gene
homologous recombination in ES cells
target vector with start codon of endogenous gene (in tissue you want) that then runs into cre. NRG too. Electroporate into ES cells
mice will have 1 normal copy gene and 1 with cre, under regulation of endogenous gene
Cre/Lox: Tet-Off System
cre gene on the tetracycline responsive promotor, tet-operon. for tetO to work it must be bound to tet activator protein.
if give mouse tetracycline, it will bind to tet-A so tetO cant be activated.
when withdraw tetracycline, tet-A is free to bind tetO –> cre will be transcribed –> floxed gene knocked out
tamoxifen induction of cre expression
oestrogen receptor is a DNA binding molecule in presence of oestrogen. it can be mutated to bind to tamoxifen.
this system uses a fusion protein, combining activity of cre and the mutant ER
in absence of tamoxifen, cre-ER will stay in cytoplasm bound to HSP90 –> won’t come into contact with floxed gene in nucleus
when give tamoxifen, the HSP90 is displaced and the complex moves to the nucleus –> floxed gene is knocked out
what is cas9
double stranded endonuclease from strep
when infected by a phage, the bacteria chops up bits of the viral genome and integrate it into sites of their genome so can target cas9 cutting enzyme to chop it up
keep memory in clustered regulatory interspaced short palindromic repeats (CRISPR)
what targets Cas9 to specific DNA sites
short RNAs
CRISPR-RNA binds to site of genome and transactivating-RNA binds to cr-RNA
complementary to target sequence
in lab can be used to cut genomic DNA of any gene which will be mutated upon repair
protospacer-adjecent motif (PAM)
cas9 only cuts upstream of specific sequence for PAM
for Cas9 it is anything then GG
how is cas9/crispr used in lab
DNA construct introduced to cells - promotor, cas9, sgRNA
sgRNA: crRNA - tracrRNA fusion
cas9-sgRNA will bind to selected bit of genome and create double stranded cut in cells
how to engineer specific mutation with cas9/crispr system
catalyse homology-directed repair
electroporate/inject cells with cas9-sgRNA-single DNA oligonucleotide homologous to the area
can encode any mutation in the oliginucleotide between homologies and the DNA repair enzymes will use it as a template to reapair the DNA
method of animal cloning
take cell from donor animal being cloned and take out nucleus
take pronucleus out recipient unfertilised oocyte
put nucleus of donor cell into enucleated oocyte
activate oocyte (chemical, electrical) to start development
complete development in utero as normal
