Medical Genomics Summary Lecture 8 Genome manipulation
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Course
Medical Genomics (AB_1143)
Institution
Vrije Universiteit Amsterdam (VU)
Orderly and clear summary of lecture 8 Genome manipulation from the book "Medical Genomics in biomedical sciences by Sander Groffen". All the summaries of the lectures match with this book therefore you will easily pass your exam. This will save you a lot of time. I passed this course with an 7,3. ...
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Vrije Universiteit Amsterdam (VU)
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Medical Genomics (AB_1143)
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Medical Genomics – Lecture 8 – Genome manipulation
DNA repair mechanisms;
Double stranded DNA is broken.
• Homologous recombination (cross-over); use sister chromatid
for accurate repair
• Non-homologous recombination (non-cross-over); if sister
chromatid is unavailable
Reverse genetics; select a gene --> find the phenotype
• Transgenesis; put a gene into the
genome of the organism, randomly
Transgene (inserted gene) consist of
promotor, ORF and poly(A)tail
Position effect; activity of the promotor is
influenced by the landing site.
In embryo’s; DNA is injected into fertilized oocyte in 1 of
the 2 pronuclei
In worms; DNA is injected into the cytoplasm of the
gonads. DNA, has to be transported to the nuclei of the
germ cells by mechanism.
This integrates randomly in the chromosome. The
transgene carries a promotor. Later on, the oocytes divide.
The injecting occurs outside the organism.
When they are born they are transgene-positive referred to founder mice.
• Homologous gene targeting; Modification of a selected locus and possibility to
eliminate endogenous genes
Specific chromosomal region is replaced by designed piece of DNA through
homologous recombination (exchange 2 identical strands)
Use of targeting vector, contain identical DNA segment to that of the gene so
recombination can occur.
Integration locus is known
Effect of gene replacement;
o Knock-out; null allele –> elimination of
endogenous gene (gene occurs in wildtype
allele)
o Knock in; mutant allele
o Conditional knock out; floxed allele
3 steps;
1. Production of targeted embryonic stem cells (ES)
ES;
They are pluripotent; can differentiate to all cell
types
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