Why do we care about homologous recombination? Universal biological mechanism Bacteria can pick up new genes Biotechnology
Gene knockouts in mice via homologous recombination
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DNA of interest in mouse chromosome
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This is the gene targeted for replacement by an engineered construct. Note flanking upstream and downstream DNA sequences. The arrows pointing away from the targeted gene represent the continuous chromosomal DNA
1. Prepare construct DNA in lab with selectable marker
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Engineered construct used to replace the gene. Upstream and downstream flanking DNA sequences are identical to those which flank the targeted gene.
2. Add construct to embryonic stem cells (ES) in culture
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Amazingly, the DNA construct finds its way into ES cell nucleus and aligns itself with targeted gene.
3. homologous recombination by cell
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The chromosome now contains engineered construct in place of the original allele. The original allele has been recombined into the construct and is lost over time.
4. Add ES cells to embryo implant in surrogate mother
5. Cross breed to create homozygous knockout
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Back to bacteria…..
Hfr strains led to mapping of the E. coli chromosome
Interrupted mating technique to map genes on E. coli
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Lederberg’s experiment explained
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Fig. 15.7
Hfr H (aziRtonRlac+gal+strS) X
F- (aziStonSlac-gal-strR)
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Circular chromosome
4.6 million bp (4.6 Mb)
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2. Transformation
Naked DNA enters bacterial cell. Brings new genes (can change bacteria phenotype)
Bacterium with new DNA is a transformant
Transformation (rare event)
Natural flash animation Engineered
CaCl2 treat bacteria competent cells cell membrane permeable to naked DNA
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Plasmids can be cloning vectors Ch 8 pg 175
pUC19
ampr geneorirestriction sites(multiple cloning site)
Plasmid requirements in biotech
1. Ori for DNA replication
2. Selectable marker ex. ampr
1. Only cells that take up the plasmid are resistant to amp
3. Restriction enzyme sites
4. High copy number in E. coli (100/cell)
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Ampr
Ori
araC
GFP
1.Shimomura
Viruses can bring new genes into a cell
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Transduction –phage mediated transfer of genes into bacteria
Bacteriophage – virus that infects bacteria
Lederberg and Zinder 1952
phage
DNA or RNA surrounded by protein coat genes encode for viral activity, viral parts
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Viral infection lytic cycle
1. Virus adsorbs to cell and injects DNA
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2. normal bacterial activity is shut down and bacterium becomes a “phage factory”
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3. host DNA broken into pieces, new viruses released to infect new cells
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chromosomal DNA is chopped as viruses destroy cell
Generalized transduction
A piece of chromosomal DNA gets packaged into a virus = faulty head stuffing
This transducing phage infects a new cell and transfers genes from the first bacterium
Homologous recombination occurs
Flash animation generalized transduction25
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Bacteriophage phenotypes
virulent phage - always lytic, cannot become a prophage
temperate phage - lysogenic
Temperate phage and lysogenic pathway
Phage DNA integrated into specific location in chromosome
Prophage is lysogenic Phage gene represses
lytic cycle
Flash animation specialized transduction
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Growing phage
Grow bacterial lawn on agar dish Add phage infects bacteria Obtain plaques (where cells have lysed) Obtain phage lysate (contains phage)
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plaques
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Gene therapy with virus (Ch 10)
Objective : insert normal gene into human DNA Candidates: people with single gene disorders Use virus as vector
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Bioinformation video
Adenovirus. Child Health and Human Development
Gene Therapy ADA 1990
Gene for adenosine deaminase ADA normally eliminates deoxyadenosine
(from degraded DNA) (recessive disease) dA toxic to lymphocytes
Severe immune deficiency
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Ashanti Disilva was 4 and dying
1. remove viral replication genes
2. insert normal ADA gene into virus
3. remove T cells from patient
4. infect cells with engineered virus
5. infuse into patient
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Problems with gene therapy
Inflammatory response to virus death Gene disrupts cell cycle gene cancer
Other methods Liposomes Stem cells
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