Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174

Biotechnology - use of living organisms to create products or help processes

Ex. HGH, insulin

Recombinant DNA - segment of DNA containing sequences from different organisms

How is DNA manipulated?

A A

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T T

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Restriction enzymes cut DNA at specific sites and create sticky ends

Complementary ends will fuse to produce a long strand of DNA

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings

• The DNA is then integrated into the recipient cell’s chromosome

Figure 12.1D

Donated DNA

Recipient cell’schromosome

Crossovers

Degraded DNA

Recombinantchromosome

bacterium

bacterialchromosome

plasmid

Plasmids are extra rings of DNA that replicate in bacteria.DNA can be inserted into plasmids.

Cloning Vectors

1. Use restriction enzymes.

2. Insert gene into plasmid.

3. Transfer the plasmid backinto bacterial cell.

4. Let bacterial cells replicate.

bacterialclones

replication

transformation

recombinant DNA

Plasmid

BacteriumHuman cell

DNA

Human proteinBacterialchromosome


Recombinant DNA products

• “seed” protein for artificial snow

• Insulin for diabetes treatment

• Enzymes that clean up toxic waste spills

• Growth Hormones (Human, Bovine)

• TPA: Tissue Plasminogen Activator for treatment of heart attacks


• The polymerase chain reaction (PCR) can quickly clone a small sample of DNA in a test tube

• Selection of specific sequence

Figure 12.12

InitialDNAsegment

1 2 4 8

Number of DNA molecules



• Restriction fragments of DNA are compared by size

Gel electrophoresis sorts DNA molecules by size

Figure 12.10

Mixture of DNAmolecules ofdifferent sizes

Powersource

Gel

Glassplates

Longermolecules

Shortermolecules

Completed gel

DNA forensics

Egg manipulation via microinjection.

Credit: © Science VU/Visuals Unlimited

Egg microinjection to produce transgenic animal

Grow bigger fish faster. Salmon with gene from another fish species


• Uses of transformed animals:

• Produce medicines more easily

Ex. sheep and gene to treat cystic fibrosis

Goats and AT3 gene to prevent blood clots

Figure 12.16

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings Fig. 11-14, p.173

An extremely large Agrobacterium tumefaciens tumor (crown gall disease) and secondary tumors on Kalanchoe stem.

Credit: © Brad Mogen

Genetic engineering of plants

Methods to insert DNA:

1. Ballistics2. Protoplasts3. Agrobacterium as vector

- Ti plasmid

a A bacterial cell contains a Ti plasmid (purple) that has a foreign gene (blue).

b The bacterium infects a plant and transfers the Ti plasmid into it. The plasmid DNA becomes integrated into one of the plant’s chromosomes.

c The plant cell divides. Its descendant cells form an embryo, which may develop into a mature plant that can express the foreign gene.

A young plant

expressing a fluorescent gene product

Fig. 11-12, p.171


Genetically modified crops

• Golden rice with Vitamin A

• Cotton resistant to boll weevil

• Soybeans resistant to herbicide (Roundup)

• Corn resistant to European corn borer

• Rapeseed with healthier vegetable oil

Benefits and risks

white sheep

black sheep

egg cell

udder cells

DNA

embryo

Dollysurrogatemother

How Dolly was cloned

Cloning of human cells

• Regenerative medicine– Bone, pancreas cells, skin

Stem cells - the $6 billion promise?


• treat disease by altering an afflicted individual’s genes

– Ex vivo

– In vivo

– Stem cells

Gene therapy may someday help treat a variety of diseases

Figure 12.19

Cloned gene (normal allele)

1 Insertnormal geneinto virus

Viral nucleicacid

Retrovirus

2 Infect bonemarrow cellwith virus

3 Viral DNAinserts intochromosome

Bone marrowcell from patient

Bone marrow

4 Inject cellsinto patient

Human Genome Project

• 3.2 billion bases in 22 autosomes + X, Y

• Draft sequence completed in 2003

• Available at www.ornl.gov/sci/techresources/Human_Genome/home.shtml

• www.ucsc.genome.edu

What does the human genome sequence tell us?• 20 K to 25 K genes• 99.9% alike, across all races• 97% of DNA is not transcribed

- Spacers between genes- Structural (centromeres, telomeres)

- Regulatory (enhancers, promoters)

- Leftovers of evolution?


How are specific genes identified?

1. Isolate it from a genomic library by homology with a gene from another organism.

2.Find mRNA for the gene, make cDNA from it.

3.Make DNA sequence based on protein sequence.


1. Nucleic acid probes identify clones carrying specific genes

Figure 12.8A

Radioactiveprobe (DNA)

Mix with single-stranded DNA fromvarious bacterial(or phage) clones

Single-strandedDNA

Base pairingindicates thegene of interest

• A nucleic acid probe can tag a desired gene in a library

Fig. 11-4, p.164

mRNA

mRNA

cDNA

DNA

DNA

reversetranscriptase

DNApolymerase

cDNA

• Complementary DNA

• Using reverse transcriptase

• Assembles DNA on mRNA template


• A labeled probe can reveal patterns of gene expression in different kinds of cells

DNA microarrays test for the expression of many genes at once

Figure 12.9

cDNA

DNA of gene

DNAmicroarray,actual size(6,400 genes)


Gene Therapy

• What is it?

• How is it done?

• Does it work?


Gene therapy

• Goal - Treat diseases caused by mutated genes

• Method - Add a normal gene or block an abnormal gene in enough cells to restore normal function

• Target - somatic cells


Which disorders are candidates for gene therapy treatment?

• Disorders due to mutations in one or more genes

• The responsible gene is known

• The affected tissues are known and accessible


Knockout gene therapy

• Goal: turn off a gene that is causing a disorder

• Strategies:

– Antisense

– Triple helix oligos

– Spliceosome

– Ribozyme


How is gene therapy done?

1. Identify the gene(s) responsible for the disorder

2. Make copies of the normal gene

3. Insert the copies into vectors (i.e., viruses)

4. Infect the affected cells with the vectors

5. Activate the gene

– Transcription and translation take place


Critical factors in choosing a vector

Gene size

– Limited room in vector genome

• Target tissue

– What cells can the vector infect?

• Integration into the genome

– Without integration, only short-term effect

– Random integration may disrupt other genes

• Cell cycle stage (dividing vs. non-dividing)


Gene Therapy Successes

Ashanti de Silva successfully treated for ADA deficiency - 1990

Ryes Evans successfully treated for SCID - 2001

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Gene Therapy Problems

Jesse Gelsinger died of complications due to an immune system response while participating in a clinical trial

Three children treated for SCID developed leukemia due to disruption of a gene that regulates cell division


Ethical and Social Issues

• Patient safety while participating in clinical trials

• Which applications are therapies and which are enhancements?

– “Designer” babies

• Access to gene therapies

Documents

Copyright © 2003 Pearson Education, Inc. publishing as Benjamin Cummings p.174