BCH3031 Lecture Notes - Lecture 15: Microinjection, Hypoxanthine-Guanine Phosphoribosyltransferase, Gene Knockout
25 May 2018
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Lecture 15 – Studying Gene and Protein Function
Modelling Genetic Mutations
• Model Organisms used for humans
o Yeast (Saccharomyces cerevisiae)
▪ Single cell eukaryote
▪ 6000 genes, easily cultured
▪ Widely used
▪ 20% human disease genes have yeast homologues
▪ Cellular processes: DNA repairs
o Worm (C.elegans)
▪ 959 somatic cells
▪ Good development/neural model, cell lineage studies
▪ Expression studies, GFP-linked gene
▪ Gene knockout by RNAi technology
o Fruit fly (Dresophila)
▪ Large polytene chromosomes, easy manipulated P-element
▪ Good developmental mode, single mutants (reverse genetics
approach)
▪ Good homology to human gene families
o Zebra fish
▪ Good model of vertebrate development
▪ Short generation/1000 offspring, transparent embryo
▪ Mutation screen – ethylnitrosourea (ENU)
▪ Genetic and physical maps improving
o Rodents – mice and rats
▪ Widely used model of human disease
▪ Genome size, no. of genes similar
▪ Patterns of embryonic development same/similar
▪ Short generation time, culture cells in vitro
▪ Some redundant or altered biochemical pathways
• Lesch-Nyans disease – HPRT gene
• If knock this gene out in mouse – still functional
▪ Absent or altered developmental pathway (in brain folding)
▪ Some genes are lost, some are gained – different functions
o Sheep and pigs
o Non-human primates
• Molecular Genetic Techniques: Gene targeting, CRISPR/Cas9 Gene editing
o Transgenic mice (adding genes): pronuclear injection
▪ Random injection of
Inj. DNA, single
site, normally as
multimer
▪ Expression of
transgene dependent
on promoter
▪ Used in mice and
livestock – drug,
protein production
find more resources at oneclass.com
find more resources at oneclass.com
Document Summary
Lecture 15 studying gene and protein function. Dna, single site, normally as multimer: expression of transgene dependent on promoter, used in mice and livestock drug, protein production. Gene targeted mice: homologous recombination in es cells. Insert dna in inner cell mass of blastocyst: targeted insertion of vector dna by homologous recombination, culture cells in eomysin, producing a mouse containing the targeted gene knockout, one mutant, one normal chromosome (single amino acid change) Nr3c2 mineralocorticoid receptor gene dimerization mutant": disrupt structure of finger receptor doesn"t bind to dna anymore, inducible knockouts deoxycyclin/tamoxifen, doxycycline-inducible cre/loxp recombination system: generation of. Grspc-cremice: taxoxifen: er(lbd)/cre-recombinase, spc promoter drives transactivator protein. *embryonic stem cells are derived from inner cell mass of a blastocytst. Correcting genetic disease/ipsc: embryonic stem cells, mouse es cells can be directed to differentiate into neural stem cells and functional glial cells and neurons.
