Lecture 5

Lecture 5 - Homologous recombination Genetic Tool and...

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Homologous recombination Genetic Tool and Meiosis Suggested scientific papers within the lecture
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Summary • Concepts – Embryonic Stem Cells – Targeting Vector – Prophase I of meiosis – Synaptonemal complex – Programmed Double strand breaks – Crossing over
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Homologous recombination as a genetic tool A series of discoveries over the last 50 years in two independent fields generated one of the most power genetic tools for discovering gene function in mammals 1) The ability to grow stem cells that can incorporate into any cell of the body 2) The ability to induce homologus recombination using recombinant DNA in the lab
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ESCs are pluripotent and are derived from the inner cell mass - (Left) Blastocyst at the stage of ESC derivation - In mice this is Embryonic day E3.5 or ~72-84 hours after fertilization - Arrow is pointing to the inner cell mass of the blastocyst
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Mouse ESCs are injected into blastocysts Single cells 12-15 cells are injected Blastocysts are implanted into the uterus
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Mice born following blastocyst injections of ESCs ESCs were derived from an agouti blastocyst (brown) and injected into a blastocyst from a black mouse
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If ESCs differentiate into germ cells this will propagate DNA into future generation
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Homologous recombination requires a vector Genomic DNA Genomic DNA Antibiotic Resistance 5’ 3’ HSV-TK Basic targeting vector (Double stranded DNA)
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Targeting the HPRT gene in embryonic stem cells Mario Capecchi Oliver Smithies
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Homologous recombination verses random integration
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Targeting vector in papers Sugo et al., EMBO 2000, 19:1397-1404
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Recombinase is used to genetically engineer a targeted locus after homologous recombination Removes pieces of DNA inserted into the genome by homologous recombination The integrase family of DNA recombinases has over 60 members Cre recombinase is the most familiar family member Cre catalyzes site-specific recombination between two, 34-base pair lox DNA sequences
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Locus X-over P1 (LoxP) 13bp 8bp 13bp ATAACTTCGTATA - GCATACAT -TATACGAAGTTAT X2 palindromes and a central 8bp central sequence
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The direction of the Lox site is important • Two LoxP sites on the same chromosome in the same direction will cause an inversion • Two LoxP sites on the same chromosome in opposite directions will cause a deletion
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Removing the neomycin resistance gene with Cre is now standard as neo has been found to cause splicing mutations Nagy et al. Current Biology 1998: 8: 661-664
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Summary • Homologous recombination used to repair double strand breaks in DNA has a an new role in the molecular biologists tool box • Homologous recombination to insert a targeting vector into the genome requires exchange of genetic material between the DNA and the vector together with DNA polymerization and ligation
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Homologous recombination in meiosis
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Sexual Reproduction
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Why is meiosis important? One set of chromosomes is obtained from each parent
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This note was uploaded on 03/30/2009 for the course MCDB 144 taught by Professor Dr.clarkanddr.lowry during the Spring '09 term at UCLA.

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Lecture 5 - Homologous recombination Genetic Tool and...

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