Lecture notes from Imperial College London, Medical Biosciences BSc, 2nd year, genetics and genomics (GEN) module.
Description of genome engineering methods, particularly in mammals, and the principles underlying them. Nowadays, we can now modify genotypes of most organisms directly, quickly and...
Engineering the Genome
making define change to the genome
Bacterial transformation
- bacterial transformation: exogenous DNA used to alter the genome of bacteria
=> (natural)
-
- incorporation by homologous recombination (HR)
=> DNA incorporated must be homologous to the
recipient genome (closely related)
=> plasmid replication origin must be inactivated
- competent bacteria: capable of taking up DNA
=> naturally (take up linear single stranded plasmid)
=> artificially (take up circular plasmids)
=
=> bacteria reproduce by binary fission => no diversity => bacterial transformation
Genome engineering in bacteria
- genomic DNA was used to transfer a characteristic from 1 strain to another
=> gene targeting: transfer of DNA into a cell to make a defined genomic modif using HR
=> selectable marker genes (antibiotic resistance...): modify genomic loci of unknown function
linearised plasmid with regions of homology (b,
c) + a marker gene (red)
=> 2 crossovers: homology sequence replaced
=> cells can then be selected in antibiotic
Rq: replacement & insertion
- single nucleotide change using an insertion constructs (plasmid):
construct are types of
targeting constructs
- Step 2: second crossover => excision of
insertion construct => subtle mutation left
Genome engineering in mammalian cells
- mammalians generate diversity with meiotic HR (don’t need exogenous DNA)
- transfection = introduce DNA artificially in mammalian cells (transformation for mammalians):
Injection with a micro-needle
DNA into viral particules
=> efficient
=> efficient U => need time + skill
=> time consuming
Capacitor induces transient + lipids & - charged DNA
membrane pores endocytosed by cells
=> large cell number => simple
=> need special equipment => no large cell number
- unlike bacteria + yeast, mammalian cells integrate DNA into chr at random sites (no homology)
=> NHEJ: error prone => incorporate exogenous DNA at DSBs
=> HR integration occur but 10 to 1000-fold less than random integration
=> only at homologous sites
=> inactive in G1 protein mRNA NHEJ
- fate of transferred DNA:
HR
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