Genetic manipulation 2 Flashcards
MsX1 deficient mice
cleft palate and craniofacial/tooth abnormalities
2 licenses required for genetically modifying animals
project license
personal license
Act for project license when working with GM animals
Animals (scientific procedure act) 1986
4 reasons for genetic manipulation
to understand genetic basis of human health and disease
To identify and analyse roles of genes - over expression and knockout
understand control of gene regulation
genetically tag animals/designer animals for disease models
3 examples of spontaneous mouse mutations
small eye - Pax 6
looptail - vangl2
clubfoot - limk1
Random mutagenesis - how is it done?
male subjected to mutagen and leads to randomly distributed point mutations at low frequency through genome
examples of agents used in random mutagenesis
radiation
ENU
EMS
ENU - what does it do?
creates point mutations by ethylating DNA base pairs in replicating sperm
EMS - what does it do?
turns G/C into A/T
screening for dominant mutations
male mated with wild type female
heterozygous = phenotype
screening for recessive mutations
may not have a phenotype if heterozygous - breed litter of progeny then mate brothers and sisters
uses of random mutagenesis
can generate mutations in tissues without prior assumption of important genes
generate new alleles of genes never made deliberately or thought of
disadvantages of random mutagenesis
use large numbers of animals
wasteful - increasingly hard to justify
basis of gene knockouts
delete gene in ES cells and inject into blastocyst
homologous recombination - when does it usually occur?
meiosis
briefly explain what homologous recombination is
identical sequences on maternal and paternal chromosomes line up and cross over
what can homologous recombination be used for?
to introduce new DNA into cells
Important note about flanking DNA sequences
transgene has identical flanking DNA to sequence that normally surrounds DNA
What can you replace the gene with in homologous recombination to make it more recognisable?
fluorescent green protein
neomyicin resistance
After electroporating cells explain how homologous recombination will work
Over the next couple of days, most ES cells will expel or degrade any DNA they took up
A small proportion will integrate targeting vector into their genome randomly
A small proportion of those cells that integrate targeting vector will do so by Homologous Recombination
After homologous recombination what ES cells should be selected?
ganciclovir - cells with TK are killed
neomycin - cells without neoR are killed
after selecting ES cells post homologous recombination what is done next?
PCR and southern blotting on genomic DNA from survivors to check integrity of integrated vector
What % of integration events does hr account for?
<2%
1 in ? cells undergo homologous recombination
10 million
1 in ? cells come through +ve/-Ve selection will turn out to be correctly targetted
200
Give an example where redundancy may lead to mild or no phenotype
myoD
Example of early embryonic lethality preventing analysis of later events
oct 4 or fgf 4
what else may affect the interpretation of knock outs?
genetic background and strain of mouse
knock in
the use of targeting vector and homologous recombination to introduce a new functional gene at a known location in a genome
use of knock ins
reporter mice expressing Green fluorescent protein
expressing one gene under control of a promoter of a different gene
therapeutics from animals - gene knock in
b - interferon = important in therapeutic
green fluorescent milk…
