Lecture #21, Biology 115: Biotechnology

T. Irving 1/21/99; revised A.Howard 04/09/00

Goals of this Section:

Students should be able to:

• List and describe the means by which cells are transformed by genetic engineering
• List and give examples of biotechnology products and biotechnology techniques
• Discuss the importance of transgenic plants, bacteria, and animals
• Describe possible methods of gene therapy in humans

Biotechnology includes:

• Recombinant DNA and related techniques e.g. immunology
• Production of drugs, vaccines, enzymes and chemicals for industry
• Production of improved crops and farm animals
• Gene therapy for human genetic diseases
• Determining the sequence of the human genome
• Immunotherapy for human diseases
• Detection of human diseases esp. cancer
• Forensic (crime detection) applications

Basics of Recombinant DNA

First stages are done in vitro

1. Isolate a gene of interest from the genome of some host or synthesize from nucleotides using a "gene assembler" machine or use polymerase chain reaction (PCR)
2. Insert into a vector, a self replicating DNA molecule to form a recombinant DNA molecule.
3. Place recombinant DNA molecule into a host cell for various purposes. Subsequent operations are said to be in vivo

• The gene of interest will confer some useful property on the host cell.
• The new cell is said to be genetically engineered or a recombinant organism.
• The gene of interest must be selected with a certain purpose is mind and then isolated or synthesized. This is not always easy to do.

Tools of Genetic engineering

• Restriction enzymes
  Cuts DNA at specific sites identified by short DNA sequences
• Ligases
  Rejoins cut ends of DNA
• Vectors
  Frequently are plasmids, small circular pieces of DNA that are readily taken up by bacteria. A number of viruses (e.g. bacteriophages) can also be used as vectors. Animal cells can be microinjected directly with DNA without a vector intermediate.
• Gel electrophoresis
  DNA restriction fragments can be separated on the basis of size on agarose gels
• Probes
  Short sequences of DNA that are complementary to the piece of DNA you want to locate. Most often have radioactive atoms attached so that they can be located by autoradiography
• Autoradiography
  Expose a piece of X-ray film by placing it next to a gel that has radioactive probes attached. The position of probes can be identified by the location of spots on the film
• Gene sequencing
  Determining the order of nucleotides in a gene
• DNA synthesis
  Producing synthetic sequences of DNA. Can be done with automated machines

Genetically engineered bacteria

Recombinant DNA molecule is manufactured in vitro containing a particular gene of interest (e.g. human insulin, growth hormone, HIV glycoprotein for vaccine etc.)

• Recombinant molecule is inserted into a bacterium
• Bacteria now effectively a factory to produce protein
• List of proteins manufactured this way for drugs and vaccines is long and getting longer
• Other products are enzymes and chemicals for industrial use (much activity by IIT Biology faculty in this area)
• An important class of products are those for bioremediation
• Engineer bacteria to degrade or eat pollutants like crude oil
• Dr. Webster at IIT and Dr. J. Kilbane of IGT head such a project to remove sulfur from coal using genetically engineered bacteria. Dr. Stark and Dr. Cork also have bioremediation projects

Agricultural Applications

Same things that can be done with bacteria can also be done with plants but need to use special plant specific vectors such as those based on T_i plasmid

• Single cells of a desired plant are isolated
• Recombinant DNA is introduced into the cell's genome by :
  • coculture with bacteria containing the recombinant DNA.
  • Electroporation i.e. use an electric field to move DNA into the host
  • Shooting host cells coated with tiny bullets coated with naked DNA
    (improbable as it may seem, it works!)
  • The now genetically engineered single cell is regenerated into a plant
  • All cells of this plant will be genetically engineered
• Beneficial Effects of Genetic Engineering on Plants
  Possible genes of interest might include:
  • Those that confer resistance to insect or fungal pests
  • Improve resistance to drought
  • Eliminate dependence on certain fertilizers
  • Improve the nutritional value of the plant
  • Produce plants that produce valuable chemical or even drugs
  • First transgenic plant, a tomato went on the market in 1994
  • Since then cotton, corn, wheat have been made more resistant to insect infestations using recombinant DNA technology
• Farm Animals & Pharm Animals
  • People have been genetically engineering domesticated animals the old fashioned way for millennia.
  • Aim is to improve meat, size, milk production, growth rate to market etc.
  • Genetic engineering techniques could vastly accelerate this process
  • Recently sheep and cows have been genetically engineered to make certain human drugs more efficiently than can be done with bacteria.
  • Gene of interest ---> animal egg cell ---> in vitro fertilization (IVF) ----> animal has all cells carrying the gene of interest
  • Usually used to insert genes for certain human hormones.
  • Hormones can be harvested from the animals milk
  • Pharmaceuticals ---> Pharm Animals

Gene Therapy

• Genetic Engineering of humans is called gene therapy because it is generally used to cure genetic diseases (there are 1000's of these).
• In principle could genetically engineer any desirable trait but ethically unacceptable
• The gene of interest is now a good or non-mutated form of the mutant gene that causes the disease
• Disease is typically only diagnosed after baby is born
• Cannot deliver rec DNA to human egg or sperm in order to ensure that all cells have the gene
• So delivery of recombinant DNA is complicated.
• Now can only be done with cells, like white blood cells, that can be removed from the body, engineered and cultured in vitro, and then returned to the body.
• Called Ex vivo gene therapy.
• First actual case of gene therapy
  • Deficiency of the protein Adenosine deaminase (ADA) causes collapse of the immune system
  • Remove stem cells or WBC's by taking blood samples
  • Culture cells & genetically engineer with recombinant DNA containing good ADA gene using a retroviral vector
  • Reintroduce cultured cells into host (transfusion)
  • New stem cells and /or WBC's make ADA and immune system recovers.
  • This procedure to recover function of ADA only worked because blood cells are easy to remove and to reintroduce
• Gene therapy of other genetic diseases with require delivery of rec DNA to many different cell types, most of which cannot be easily removed and reintroduced
• Will require new technological advances;
  e.g. recent trial in which good cystic fibrosis gene is introduced via a recombinant adenovirus via an aerosol spray

Strategies using genetic engineering to combat cancer

• Remove tumor cells from body
• Insert gene for interleukin-2 (IL-2): a cytokine that helps immune system attack tumor cells
• Reintroduce to the body
• Strong immune response may enhance attack on non-engineered cells as well
• Insert good proto-oncogenes into tumor cells to counteract effects of mutated versions
• e.g. p53, a tumor suppressor gene

Human Genome Project

• Large NIH funded project involving many labs all over the country
• Uses recombinant DNA and related techniques
• Idea is to map & eventually determine base sequence of all human genes
• Will probably be of tremendous importance in diagnosis of genetic diseases
• Will also aid in determining (at a very detailed level) causes of these diseases so that systematic gene and drug therapies can be devised

Polymerase Chain Reaction (PCR)

• Usually only tiny amounts of DNA can be isolated from individual living organisms, too little for engineering purposes
• DNA can be amplified by cloning i.e. adding recombinant DNA to bacteria and grow up many kg of bacteria
• A more recent technique is the Polymerase Chain Reaction or PCR
• Millions of copies of a DNA fragment can be made in a few hours entirely in vitro.
• Uses DNA polymerase, the enzyme normally used by bacterial cells to make copies of DNA in the cell cycle
• Can amplify tiny amounts of DNA from fossils, embryonic cells, tissue or semen samples from crime victims.
• Jurassic Park posits the use of PCR on a grand scale: dinosaur DNA to make dinosaurs

DNA fingerprinting

• Genes exist in different forms called alleles
• When cellular DNA is cut with restriction enzymes they form restriction fragments of different sizes depending on the alleles present.
• Different sized fragments when separated on a gel show restriction fragment length polymorphisms (RFLPs)
• Since each individual has different alleles for their genes, each individual will show a characteristic DNA fingerprint.
• Hasn't always been acceptable as a form of proof of guilt in criminal investigations but is very useful in eliminating suspects.
• Has been used to identify rapists and murderers and in some cases to release innocent persons from prison.

Ethics, Safety and Genetic Engineering

• In early days people were very concerned that gene-splicing projects might create (accidentally or deliberately!) a super-bacterium that could escape and cause widespread incurable diseases
• Recombinant research, for the most part, uses organisms that cannot live outside the laboratory.
• We know that bacteria promiscuously exchange DNA and accidents are inevitable.
• Other concerns are ecological catastrophes due to genetically engineered plants or other organisms could take over from native plants if introduced into the wild.

Many ethical questions concerning genetic engineering on humans

• When and on whom should genetic testing be done?
• Who should have access to results of genetic testing?
• Should people be denied employment or insurance because they have genetic profiles deemed defective?
• With spiraling medical costs, should carriers of genetic disorders be denied the right to have children? Forced to pay high insurance premiums?
• Do humans have the right to produce and patent new transgenic species?
• What about medical researchers patenting genetic material from cells derived from your body with or without your consent?