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

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

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 1. Prepare construct DNA in lab with
selectable marker

Engineered construct used to replace the gene.

Upstream and downstream flanking DNA sequences
are identical to those which flank the targeted gene.

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 2. Add construct to embryonic stem cells
(ES) in culture

Amazingly, the DNA construct finds its way into ES

cell nucleus and aligns itself with targeted gene.

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 3. homologous recombination by cell

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.

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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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Lederbergs experiment explained

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 Fig. 15.7

Hfr H (aziRtonRlac+gal+strS)

F- (aziStonSlac-gal-strR) 9
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

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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 gene
ori
restriction sites
(multiple cloning site)

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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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1.Shimomura

Ampr

Ori

araC

GFP

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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

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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

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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 transduction

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Bacteriophage phenotypes
virulent phage - always lytic, cannot
become a prophage

temperate phage - lysogenic

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 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

Adenovirus. Child Health and

Human Development

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Bioinformation video
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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