1. Lecture 11 Biotechnology

2. A Scientific Revolution  Genetic engineering is the process of moving genes from one organism to another  Having a major impact on agriculture & medicine

3. Restriction Enzymes  Restriction enzymes bind to specific short sequences (usually 4- to 6- bases long) on the DNA  The nucleotide sequence on both DNA strands is identical when read in opposite directions  Most restriction enzymes cut the DNA in a staggered fashion  This generates “sticky” ends  These ends can pair with any other DNA fragment generated by the same enzyme  The pairing is aided by DNA ligase GAATTC CTTAAG Play Restriction Enzymes

4. 4 Stages of a Genetic Engineering Experiment 1.Cleaving DNA 2.Producing recombinant DNA 3.Cloning 4.Screening All gene transfer experiments share four distinct stages Play Steps in cloning a gene

5. Stage 1  Cleaving the DNA  The large number of fragments produced are separated by electrophoresis Fragments appear as bands under fluorescent light

6. Stages 2 & 3  Producing Recombinant DNA  Fragments of source DNA are inserted into vectors  Vectors are plasmids or viruses that carry foreign DNA into the host cell  Vector DNA is cut with the same enzyme as the source DNA, thus allowing the joining of the two  Cloning  Host cells are usually bacteria  As each bacterial cell reproduces, it forms a clone of cells containing the fragment-bearing vector  Together all clones constitute a clone library

7. Stage 4  Screening  A preliminary screen of the clone library eliminates 1.Clones without vectors 2.Clones with vectors that do not contain DNA  The vector employed usually has genes for  Antibiotic resistance  This eliminates the first type of clones because they are sensitive to antibiotics   -galactosidase  This eliminates the second type of clones based on X-gal metabolism and color changes

8. Stage 4 (cont.)  Screening  To find the gene of interest, the clone library is screened by a process termed hybridization  The cloned genes form base pairs with complementary sequences on another nucleic acid, termed the probe  The bacterial colonies are first grown on agar  They are then transferred to a filter  The filter is treated with a radioactive probe  The filter is then subjected to autoradiography

9. Working with DNA  Key techniques used by today’s genetic engineers include  PCR amplification  Used to increase the amounts of DNA  cDNA formation  Used to build genes from their mRNA  DNA fingerprinting  Used to identify particular individuals

10.  The polymerase chain reaction (PCR) requires primers  Short single-stranded sequences complementary to regions on either side of the DNA of interest  PCR consists of three basic steps 1.Denaturation 2.Primer annealing 3.Primer extension PCR Amplification Target sequence Primers Denaturation 1 Heat 2 Annealing of primers Cool 2 copies Free nucleotides 3 Primer extension DNA polymerase Cycle 1 Heat Cool 4 copies Cycle 2 Cool Heat 8 copies Cycle 3 Play Polymerase Chain Reaction

11.  The primary mRNA transcript contains exons and introns  The processed mRNA contains only exons  It is used as a template to create a single strand of DNA termed complementary DNA (cDNA)  cDNA is then converted to a double-stranded molecule cDNA Formation

12.  This is a process that is used to determine if two DNA samples are from the same source  The DNA from the two sources is fragmented using restriction enzymes  The fragments are separated using gel electrophoresis  They are transferred to a filter  The filters are screened with radioactive probes  Then subjected to autoradiography DNA Fingerprinting Play DNA Fingerprinting

13. Genetic Engineering and Medicine  Genetic engineering has been used in many medical applications 1.Production of proteins to treat illnesses 2.Creation of vaccines to combat infections 3.Replacement of defective genes

14.  In diabetes, the body is unable to control levels of sugar in the blood because of lack of insulin  Diabetes can be cured if the body is supplied with insulin  The gene encoding insulin has been introduced into bacteria  Other genetically engineered drugs include  Anticoagulants  Used to treat heart attack patients  Factor VIII  Used to treat hemophilia  Human growth hormone (HGH)  Used to treat dwarfism Making “Magic Bullets”

15.  Genetic engineering has also been used to create subunit vaccines against viruses Piggyback Vaccines A gene encoding a viral protein is put into the DNA of a harmless virus and injected into the body The viral protein will elicit antibody production in the animal A novel kind of vaccine was introduced in 1995 The DNA vaccine uses plasmid vectors It elicits a cellular immune response, rather than antibody production

16. Genetic Engineering of Farm Animals  In 1994, the recombinant hormone bovine somatotropin (BST) became commercially available  Dairy farmers used BST as a supplement to enhance milk production in cows  Consumers are concerned about the presence of the hormone in milk served to children  This fear has not been supported by research data

17. Genetic Engineering of Crop Plants  Pest resistance  Leads to a reduction in the use of pesticides  Bt, a protein produced by soil bacteria, is harmful to pests but not to humans  The Bt gene has been introduced into tomato plants, among others  Herbicide resistance  Crop plants have been created that are resistant to glyphosate  Herbicide resistance offers two main advantages  Leads to a reduction in the use of pesticides  Lowers the cost of producing crops  Reduces plowing and conserves the top soil

18. Genetic Engineering of Crop Plants  More Nutritious Crops  Worldwide, two major deficiencies are iron and vitamin A  Deficiencies are especially severe in developing countries where the major staple food is rice  Ingo Potrykus, a Swiss bioengineer, developed transgenic “golden” rice to solve this problem

19.  The promise of genetic engineering is very much in evidence  However, it has generated considerable controversy and protest  Are genetic engineers “playing God” by tampering with the genetic material?  Two sets of risks need to be considered 1.Are GM foods safe to eat?  The herbicide glyphosate blocks the synthesis of aromatic amino acids  Humans don’t make any aromatic amino acids, so glyphosate doesn’t hurt us  However, gene modifications that render plants resistant to glyphosate may introduce novel proteins  Moreover, introduced proteins may cause allergies in humans 2.Are GM foods safe for the environment?  Three legitimate concerns are raised 1.Will other organisms be harmed unintentionally? 2.Will pests become resistant to pesticides? 3.What if introduced genes will pass from GM crops to their wild or weedy relatives? Potential Risks of Genetically Modified (GM) Crops

20.  Should GM foods be labeled? Every serious scientific investigation has concluded that GM foods are safe So there is no health need for a GM label However, people have a right to know what is in their food So there may be a need for label after all

21. Cloning Higher Organisms  The successful embryos (about 30 in 277 tries) were transplanted into surrogate mother sheep  On July 5, 1996, “Dolly” was born  Only 1 of 277 tries succeeded  However, Wilmut proved that reproductive cloning is possible  Since Dolly, scientists have successfully cloned sheep, mice, cattle, goats and pigs  However, problems and complications arise, leading to premature death  Dolly died in 2002, having lived only half a normal sheep life span

22. Embryonic Stem Cells  The blastocyst, an early embryo, consists of  A protective outer layer that will form the placenta  Inner cell mass that will form the embryo  The inner cell mass consists of embryonic stem cells  These are pluripotent  Capable of forming the entire organism  As development proceeds, cells lose their pluripotency  They become committed to one type of tissue  They are then called adult stem cells  The research in human embryonic stem cells is associated with two serious problems  Finding a source: harvesting them from discarded embryos raises ethical issues  Immunological rejection: Implanted stem cells will likely be rejected by the immune system of the individual

23. Stem Cells  Embryonic stem cells could be used to restore tissues lost or damaged due to accident or disease  Experiments have already been tried successfully in mice  Damaged spinal neurons have been partially repaired  The course of development is broadly similar in all mammals  Therefore, the experiments in mice are very promising

24. Grappling with the Ethics of Stem Cell Research  Stem cells offer enormous promise for treating a wide range of diseases  However, the research involves ethical issues 1.Destruction of human embryos  When does human life begin? 2.Possibility of future abuse or misuse  Is human reproductive cloning next? 3.Alternative sources of stem cells  Are adult stem cells equally effective?

25. Gene Therapy  Gene therapy involves the introduction of “healthy” genes into cells that lack them  It was first used successfully in 1990  Two girls were cured of a rare blood disorder caused by a defective adenosine deaminase gene  The girls stayed healthy  In 1999, AAV successfully cured anemia in rhesus monkeys  AAV was also used to cure dogs of a hereditary disorder leading to retinal degeneration & blindness