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
The benefits of buying summaries with Stuvia:
Guaranteed quality through customer reviews
Stuvia customers have reviewed more than 700,000 summaries. This how you know that you are buying the best documents.
Quick and easy check-out
You can quickly pay through credit card or Stuvia-credit for the summaries. There is no membership needed.
Focus on what matters
Your fellow students write the study notes themselves, which is why the documents are always reliable and up-to-date. This ensures you quickly get to the core!
Frequently asked questions
What do I get when I buy this document?
You get a PDF, available immediately after your purchase. The purchased document is accessible anytime, anywhere and indefinitely through your profile.
Satisfaction guarantee: how does it work?
Our satisfaction guarantee ensures that you always find a study document that suits you well. You fill out a form, and our customer service team takes care of the rest.
Who am I buying these notes from?
Stuvia is a marketplace, so you are not buying this document from us, but from seller lisaribau. Stuvia facilitates payment to the seller.
Will I be stuck with a subscription?
No, you only buy these notes for $10.46. You're not tied to anything after your purchase.
