Michael J. Freudiger

and others, mod.

BiotechnologyRecombinant DNA and its

Applications

Biotechnology

Dolly and surrogate Mom

Genetically modified rice.

Embryonic stem cells and gene therapy

Biotechnology

Biotechnology, defined broadly, is the engineering of organisms for useful purposes.

Often, biotechnology involves the creation of hybrid genes and their introduction into organisms in which some or all of the gene is not normally present.

Fourteen month-old genetically engineered (“biotech”) salmon (left) and standard salmon (right).

Biotechnology

• The use of living cells to make products such as pharmaceuticals, foods, and beverages

• The use of organisms such as bacteria to protect the environment

• The use of DNA science for the production of products, diagnostics, and research

What Are Some Applications of Recombinant DNA Technology?

Bacteria, Yeasts, and Plants can all be modified to produce important pharmaceuticals, enriched foods, and industrial products.

We’ll examine

Animal cloning

The promise and perhaps perils of embryonic stem cells

DNA fingerprinting

Genetically modified foods and the American-European opinion divide.

Gene cloning for pharmaceutical production

Gene therapy

1 - Animal cloning

Dolly and her surrogate mother.

Why Clone Animals?

To answer questions of basic biology

Five genetically identical cloned pigs.

For herd improvement. To satisfy our desires (e.g. pet cloning).

For pharmaceutical production.

Is Animal Cloning Ethical?

The first cloned horse and her surrogate mother/genetic twin.

As with many important questions, the answer is beyond the scope of science.

The Biotechnology of Reproductive Cloning

Even under the best of circumstances, the current technology of cloning is very inefficient.

Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.

Saved by Cloning?

Some are firm believers while many view these approaches to be more of a stunt.

Note the use of a closely related species, a domestic goat, as egg donor and surrogate mother.

The Next Step?

Highly unlikely.

Attempts at human cloning are viewed very unfavorably in the scientific community.

2 - Gene cloning for pharmaceutical production

Recombinant DNA

• The manipulation and combination of DNA from two sources

• Bacterial DNA + human gene for insulin

• Plant DNA + bacterial DNA - Agrobacterium tumefaciens

• Mouse DNA + human DNA = transgenic

Recombination• Insert a foreign gene into a host Plasmid ( for example, exogenous DNA)

into the bacterial cell – transformation or transfection-organism referred to as transgenic ( eukaryote ) or recombinant( prokaryote)

• Goal – To produce many copies ( clones) of a particular gene

• Reporter gene – tags gene of interest – to identify the presence of a gene

Vectors

Plasmids VirusesParticles ( DNA coated bullets)Exogenous DNA

Characteristics of a Vector

• Can replicate independently in the host cell

• Has restriction sites in the vector- cloning region

• Has a reporter gene that will announce its presence in the host cell

• Is a small size in comparison to the host chromosome for ease of isolation

Plasmids and restriction enzymes

1) What are Plasmids?

2) How can we modify plasmids? Restriction Enzymes

3) Origins of restriction enzymes.

4) A close look at restriction enzymes.

5) Understanding plasmid diagrams.

What are Plasmids?

• Circular DNA that is used by bacteria to store their genetic information.

• Modifying plasmids to include extra genes allows for the production of new proteins.

In this Lecture…

How Can We Modify Plasmids?

1) Restriction Enzymes BamHI, HindIII, etc. Where do they come

from? How do they work? Different restriction

enzymes do different things.

2) DNA Ligase

Restriction Enzyme attached to DNA before cleavage

In this Lecture…

Origins of Restriction Enzymes

1) Bacteria produce restriction enzymes to protect against invading viral DNA/RNA.

Origins of Restriction Enzymes

2) The enzymes cut the invading DNA/RNA, rendering it harmless.

Restriction Enzymes are Enzymes That Cut DNA Only at Particular Sequences

The enzyme EcoRI cutting DNA at its recognition sequence

Different restriction enzymes have different recognition sequences.

This makes it possible to create a wide variety of different gene fragments.

Restriction enzyme animation

Restriction Enzyme in Action

1) DNA strand with EcoRI restriction site highlighted.

2) EcoRI restriction enzyme added (outline of separation about to occur).

3) Restriction fragments separate, with “sticky ends” at each edge.

Sticky Ends

Adding DNA Ligase

DNA ligase bonds sticky ends cut with the same restriction enzyme.

Sticky ends cut with different restriction enzymes will not bond together.

Why?

Because the base pair sequence of the two sticky ends will be different and not match up.

Sticky Ends

DNAs Cut by a Restriction Enzyme Can be Joined Together in New Ways

These are recombinant DNAs and they often are made of DNAs from different organisms.

Plasmid Maps Indicate Restriction Sites and Genes

Make Recombinant DNA Using Restriction Enzymes

Recombinant DNA- Example

DNA From Two Sources(Restriction Sites Labeled)

Circular DNA Linear DNA

Application of Restriction Enzymes

Adding DNA Ligase

Recombinant DNA Plasmid

• Many possible recombinant DNA plasmids can be produced, but this was the desired plasmid for the experiment.

Many Other Recombinant Possibilities

…and many more!

Plasmid DNA Insertion

DNA plasmids can be inserted into bacteria using a variety of laboratory processes.

Transgenic Colony Allowed to Grow

How Do We Get the Desired Plasmid?• Restriction fragments will

ligate randomly, producing many plasmid forms.

• Bacterial insertion would be necessary, then colony growth, and further testing to isolate bacteria with the desired plasmid.

Transformation of bacterial cells through electroporation.

Bacteria are then moved to a growth plate, and grown on selective media to “weed out” cells that have not picked up the desired plasmid.

Recombinant plasmids

Harnessing the Power of Recombinant DNA Technology – Human Insulin Production by Bacteria

Human Insulin Production by Bacteria

6) join the plasmid and human fragment

and cut with a restriction enzyme

Human Insulin Production by Bacteria

Mix the recombinant plasmid with bacteria.

Screening bacterial cells to learn which contain the human insulin gene is the hard part.

Route to the Production by Bacteria of Human Insulin

A fermentor used to grow recombinant bacteria.

This is the step when gene cloning takes place.

The single recombinant plasmid replicates within a cell.

Then the single cell with many recombinant plasmids produces trillions of like cells with recombinant plasmid – and the human insulin gene.

One cell with the recombinant plasmid

Route to the Production by Bacteria of Human Insulin

The final steps are to collect the bacteria, break open the cells, and purify the insulin protein expressed from the recombinant human insulin gene.

Route to the Production by Bacteria of Human Insulin

Overview of gene cloning.

Cloning animation

Pharming

These goats contain the human gene for a clot-dissolving protein that is produced in their milk.

Pharming is the production of pharmaceuticals in animals engineered to contain a foreign, drug-producing gene.

Pharmaceuticals• insulin for diabetics • factor VIII for males suffering from hemophilia A • factor IX for hemophilia B • human growth hormone (GH) • erythropoietin (EPO) for treating anemia • three types of interferons • several interleukins • granulocyte-macrophage colony-stimulating factor (GM-CSF) for stimulating

the bone marrow after a bone marrow transplant • tissue plasminogen activator (TPA) for dissolving blood clots • adenosine deaminase (ADA) for treating some forms of

severe combined immunodeficiency (SCID) • angiostatin and endostatin for trials as anti-cancer drugs • parathyroid hormone • hepatitis B surface antigen (HBsAg) to vaccinate against the hepatitis B

virus

3 - Embryonic stem cells

The Promise and Possible Perils of Stem Cells

The Stem Cell Concept

A stem cell is an undifferentiated, dividing cell that gives rise to a daughter cell like itself and a daughter cell that becomes a specialized cell type.

Stem Cells are Found in the Adult, but the Most Promising Types of Stem Cells for Therapy are Embryonic Stem Cells

The Inner Cell Mass is the Source of Embryonic Stem Cells

The embryo is destroyed by separating it into individual cells for the collection of ICM cells.

Some Thorny Ethical Questions

Is it ethical to harvest embryonic stem cells from the “extra” embryos created during in vitro fertilization?

Are these masses of cells a human?

Additional Potential Dilemmas – Therapeutic Cloning to Obtain Matched Embryonic Stem Cells

Cells from any source other than you or an identical twin present the problem of rejection.

If so, how can matched embryonic stem cells be obtained?

A cloned embryo of a person can be made, and embryonic stem cells harvested from these clones.

Cultured mouse embryonic stem cells.

Therapeutic Cloning

Is there any ethical difference between therapeutic and reproductive cloning?

4 - DNA, the Law, and Many Other Applications – The Technology of DNA Fingerprinting

A DNA fingerprint used in a murder case.

What are we looking at? How was it produced?

The defendant stated that the blood on his clothing was his.

DNA Fingerprinting Basics

Different individuals carry different alleles.

Most alleles useful for DNA fingerprinting differ on the basis of the number of repetitive DNA sequences they contain.

DNA Fingerprinting Basics

If DNA is cut with a restriction enzyme that recognizes sites on either side of the region that varies, DNA fragments of different sizes will be produced.

A DNA fingerprint is made by analyzing the sizes of DNA fragments produced from a number of different sites in the genome that vary in length.

The more common the length variation at a particular site and the greater the number the sites analyzed, the more informative the fingerprint.

A Site With Three Alleles Useful for DNA Fingerprinting

DNA fragments of different size will be produced by a restriction enzyme that cuts at the points shown by the arrows.

The DNA Fragments Are Separated on the Basis of Size

The technique is gel electrophoresis.

The pattern of DNA bands is compared between each sample loaded on the gel.

Gel electrophoresis animation

Possible Patterns for a Single “Gene” With Three Alleles

In a standard DNA fingerprint, about a dozen sites are analyzed, with each site having many possible alleles.

A DNA Fingerprint

When many genes are analyzed, each with many different alleles, the chance that two patterns match by coincidence is vanishingly small.

DNA detective animation

HGP fingerprinting page

5 - Genetically Modified Foods

Many of our crops in the US are genetically modified.

Should they be?

GM Crops are Here Today

Source: Pew Initiative on Food and Biotechnology, August 2004.

Methods for Plant Genetic Engineering are Well-Developed and Similar to Those for Animals

Golden Rice is Modified to be Provide a Dietary Source of Vitamin A

Worldwide, 7% of children suffer vitamin A deficiency, many of them living in regions in which rice is a staple of the diet.

Golden rice (yellow) with standard rice (white).

Genetically Modified Crops

Genetically Modified Cotton (contains a bacterial gene for pest resistance)

Standard Cotton

GMOs, Especially Outside the US, Are a Divisive Issue

Protesters at the 2000 Montreal World Trade Summit

European sentiment

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

The jury’s still out on the magnitude of GM crop’s ecological impact, but the question is debated seriously.

Current Concerns by Scientists Focus on Environmental, Not Health, Effects of GM Crops

6 - Gene Therapy

• Involves delivery of therapeutic genes into the human body to correct disease conditions created by faulty genes

• Two primary strategies:

• Ex vivo gene therapy

• In vivo gene therapy

Ex vivo gene therapy

• Cells from diseased person are removed

• Then, they are treated in lab (using techniques similar to bacterial transformation)

• Finally, they are reintroduced to the patient• More effective than in vivo

• Transfection is the introduction of DNA into animal or plant cells

In vivo gene therapy

• Introducing genes directly into tissues or organs without removing body cells

• Challenge is delivery only to intended tissues

• Viruses act as vectors for gene delivery, but some injected directly into tissue

Delivery of therapeutic genes

• Therapeutic genes often called payload

• May require long-term expression of corrective gene

• Others require rapid expression for short periods of time

Viral vectors

• Viral vectors use viral genome to carry therapeutic gene(s) and to infect human body cells

• Adenovirus (common cold)

• Adeno-Associated Virus

• Retrovirus (HIV)

• Herpes simplex virus (cold sores)

• Viruses must be engineered so that they can neither produce disease nor spread (extremely effective at infecting human cells)

Vector Transfection•Targeted gene therapy may result since some viruses infect certain body cells

•Adenoviruses infect both dividing & non-dividing cells effectively

•Adeno-Associated viruses do not cause illness in humans, can infect a wide variety of cells, & integrate 95% of time in same location

•Retroviruses are of interest because they insert DNA into the genome of host where it remains permanently (integration), but often, randomly

Vector Transfection•Herpes virus (HSV-1) strain primarily affects central nervous system (CNS)

•May help develop treatments for Alzheimer’s, Parkinson’s, and other genetic neurodegenerative diseases

•However, although viral vectors may help, most human cells are not easily transfected

Alternative delivery• Liposomes are small diameter, hollow particles made of lipid molecules

• Packaged with genes and injected into tissues

• A gene gun could also be used

• “Naked” DNA injected directly into body tissues (ex. effective in liver/muscle), but not enough cells express gene to have affect

• Artificial chromosomes may also deliver therapeutic gene•Non-protein coding DNA with therapeutic gene

• Similar construction to normal chromosomes (designed for permanent incorporation)

Curing Genetic Disease• More than 3,oo0 human genetic disease conditions are caused by single genes

• These are candidates for treatment by gene therapy

• Cystic Fibrosis

• Huntington’s disease

• Tay-Sachs

• Hemophilia

• Sickle cell disease

• Phenylketonuria (PKU)

Classic Example: Cystic Fibrosis

• Occurs with 2 defective copies of gene encoding the protein called cystic fibrosis transmembrane conductance (CFTR)

• Normally it serves as a pump at the cell membrane to move chloride ions out of the cells

• If cells can’t move chloride out, they absorb water trying to dilute the chloride in the cell• This leads to the production of THICK sticky mucus that clogs airways; ideal environment for infections (leading to pneumonia, etc…)

Treating Cystic Fibrosis

• Back clapping

• Drugs that thin mucus

• Antibiotic treatment (to fight infections)

• One form of gene therapy has helped

CF Gene Therapy

• Uses viruses and liposomes sprayed into nose & mouth

• Expensive treatment

• NOT a reliable cure yet

• Requires multiple applications (DNA doesn’t integrate)

• May not produce adequate protein

Unresolved Questions

• Can gene expression be controlled in the patient?

• What happens if normal gene is overexpressed?

• How long will the therapy last?

• What is the best vector to use?

• What is the minimum number of cells needed to infect to achieve success?