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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