What are the advantages (pros) What are the disadvantages (cons)? Is it right to create an animal like this simply to make our lives easier?

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1 Notes:

2 What are the advantages (pros) What are the disadvantages (cons)? Is it right to create an animal like this simply to make our lives easier? What is it?

3 Genetic Engineering Means making changes to DNA in order to change the way living things work. Creates new crops and farm animals. Makes bacteria that can make medicines. Can grow human body parts. Can prevent genetic diseases, change humans.

4 Altering Organisms isn t NEW, we ve been doing it for 1000 s of years It s called - Selective Breeding.

5 Selective Breeding

6 For Example: The Labradoodle Look at the following dogs - crossing a Poodle and a Labrador results in a Labradoodle What features has the Labradoodle inherited from the Labrador? What features has the Labradoodle inherited from the Poodle? + Poodle + Labrador Labradoodle

7 Labradoodle The Labradoodle is large (like the Labrador), has a fluffy coat (like the Poodle) which sheds much less than a Labrador s coat. They are also generally well tempered and good swimmers (like both its parent breeds). So the Labradoodle has favorable characteristics from both of its parent breeds. + Poodle + Labrador Labradoodle

8 Selective Breeding Selective breeding is a process used to produce different breeds of animals or varieties of plants that have useful characteristics Using selective breeding you can produce a specific offspring with useful characteristics of both parents What organisms do farmers selectively breed?

9 Selective Breeding Farmers can choose individual cows to mate in order to produce a generation of cows that will yield more milk. Farmers could also selectively breed for docile animals. Apple growers want to produce a type of apple that is tasty and resistant to disease. This can be done by crossing a variety of apple known for taste with another variety that shows strong resistance to disease. Farmers could also selectively breed for crops of a uniform height / when they are ready for harvest.

10 Another Example: Cows Suppose you wanted a variety of cow that produced a lot of milk: - Choose or select the cows in your herd that produce the most milk Only let these cows reproduce Select the offspring that produce the most milk Only let these offspring reproduce Keep repeating the process of selection and breeding until you achieve your goal

11 The key is to identify the feature you want, and only breed from the individuals that have that feature it is achieved by Select parents with the desired traits (things you want) Cross the parents (breed them) Select from the offspring Repeat (over many generations) Selective breeding is used to: - Choose characteristics of the food item required Produce a more uniform crop (size / harvest time) Extend the tolerance range of an organism

12 Perfect Pet Imagine you wanted to create your perfect dog how would you selectively breed for it? What two parents are you going to choose? What qualities do each of them have (which you want) How are you going to get these qualities in your pet?

13 However, the traits inherited are RANDOM and UNPREDICTABLE.

14 With Genetic Engineering, Traits are selected intentionally. Offspring receive specific DNA from parents. Desired results are usually obtained.

15 The simple addition, deletion, or manipulation of a single trait in an organism to create a desired change.

16 -Major tool is recombinant DNA. -Recombinant DNA (rdna) - DNA joined to other unrelated foreign DNA. -also called gene splicing. -tiny segments of a gene are taken out and replaced.

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18 How is it different from Selective Breeding? In selective breeding, we are combining the traits of two organisms randomly to get a random mixture of their genes and hoping for the results we seek. In genetic engineering, a single gene, a half page recipe in the 52-thousand-page set of recipe books, can direct the plant to make new traits or remove them

19 Transgenic Organisms: Are organisms that have been altered by genetic engineering. Genetic material changed by other than random natural breeding. Gene transfer - moving a gene from one organism to another.

20 Time for a quick poll: Raise your hand if you would eat food (plant or animal) that had been created through genetic modification.

21 Time for a quick poll II: Raise your hand if you would eat vegetables that had been created through genetic modification but NOT meat.

22 Time for a quick poll III: Raise your hand if you would eat meat that had been created through genetic modification but NOT vegetables.

23 Time for a quick poll IV: Raise your hand if you would eat both meat & vegetables that had been created through genetic modification.

24 Types of Transgenic Organisms GMO- Genetically Modified Organism. Uses genes not found in the organism to provide new traits. GEO - Genetically Enhanced Organism Uses superior versions of genes already found in the organisms genome.

25 What are Genetically Modified (GM) Foods? ( Frankenfoods?) You have probably already eaten GM foods. Some GM tomatoes, for example, have had their genes altered to stop them from going soft while they are still growing. For several years they were widely sold in tomato paste. The GM foods we eat have all been tested for safety. But some people worry about the long term effects of eating genetically modified foods.

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27 What Have I Eaten? Genetically modified (GM) foods possess specific traits such as tolerance to herbicides or resistance to insects or viruses. By most estimates, up to 70% of the processed foods at your local grocery store contain at least one ingredient that s been genetically altered Genetically modified to travel better so don t have to be picked when green better tasting! Genetically modified to reduce being eaten by insects.

28 Going Bananas? According to recent reports, the world may soon be out of bananas. Because of the starchy fruit s unique method of reproduction, it seems, banana plantations in Africa, Asia and Central America are uniquely susceptible to fungi, viruses and pests. Unless scientists can find a way to genetically enhance the banana s ability to ward off parasites, we could be bananaless in ten years. Several agroscience companies believe they can genetically engineer such an invincible banana by copying parts of the genetic codes of other fruits and instilling them into the banana.

29 Pros of Genetic Engineering: Crops Better taste and quality Less time to ripen. More nutrients, more food, and stress tolerance Improved resistance to disease, pests, and herbicides New products and growing techniques Animals Increased resistance, productivity, hardiness, and feed efficiency Better yields of meat, eggs, and milk Improved animal health and diagnostic methods Environment "Friendly" bioherbicides and bioinsecticides Conservation of soil, water, and energy Better natural waste management More efficient processing Society More food for growing populations

30 Safety Cons of Genetic Engineering Potential human health impact: allergens, transfer of antibiotic resistance markers, unknown effects Potential environmental impact: unintended transfer of transgenes through cross-pollination, loss of flora and fauna biodiversity Access and Intellectual Property Domination of world food production by a few companies Increasing dependence on Industralized nations by developing countries Ethics Violation of natural organisms' intrinsic values Tampering with nature by mixing genes among species Objections to consuming animal genes in plants and vice versa Stress for animal Labeling Not mandatory in some countries (e.g., U. States) Mixing GM crops with non-gm confounds labeling attempts

31 How can we use gen. eng. to help people? Right now, doctors are using pig hearts for transplants but there are still rejection problems. One day soon, scientists will be able to genetically engineer pigs to grow human organs for use in transplants. By inserting a gene for human insulin into an E.Coli bacterium, the E. coli will make lots of insulin, which scientists and doctors can collect and use.

32 In Minnesota researchers at the Mayo Clinic created pigs with human blood flowing through their bodies. And at Stanford University in California an experiment is being planned to create mice with human brain organization. Scientists feel that, the more humanlike the animal, the better research model it makes for testing drugs or possibly growing "spare parts," such as livers, to transplant into humans.

33 GM crops Crops can be given extra genes for new and useful characteristics. They are genetically modified (GM).What characteristics might be useful in crops? pest resistance frost resistance disease resistance herbicide resistance drought resistance longer shelf life

34 Pest-resistant crops Potatoes can be genetically modified so they are toxic to pests, such as the Colorado beetle. The gene for a powerful bacterial toxin is added to the potato plant. If the beetle tries to eat the potato plant, it is killed by the toxin.

35 Frost-resistant crops Crops can be genetically modified so they are resistant to adverse environmental conditions. For example, lettuces could be genetically modified to be resistant to frost. GM lettuce Why are some people against the development and use of GM crops? non- GM lettuce

36 Transgenic goats For example, the gene for a human antibody can be introduced into goats. Additional controlling DNA is also introduced, so the human antibody is only produced in the goat s mammary gland at a certain time. The antibody is then expressed in the goat s milk, where it can be purified and used to treat diseases.

37 Which came first? The eggs of this transgenic chicken contain a human antibody that could one day help to treat skin cancer. What advantages does this method of producing antibodies have?

38 Plants with extra vitamins Rice can be genetically modified to make beta-carotene, a substance that is converted into vitamin A in the body. The colour of the rice is an indication of how much more betacarotene it contains. The GM rice is called Golden Rice and is being developed to help fight vitamin A deficiency and blindness in developing countries.

39 What is Gene Therapy? 1. In people with cystic fibrosis, one of the genes is faulty and cannot do its job properly. 2. To fix the problem, a copy of the same gene from a healthy person is spliced into a virus. 3. The patient s lungs are infected with the virus. It delivers the working gene into the patient s cells. The cells can then make the right protein, and the patient can breathe normally. Patient s cell Patient s DNA Faulty Gene Virus DNA New working gene Patient s DNA Virus DNA with new gene

40 Will We Be Able To Cure Cancer With Gene Therapy? Cancer happens when body cells grow out of control. Scientists have found a gene called p-53 which normally keeps cells under control. They think that in some people with cancer, the disease begins because the p-53 gene doesn t work properly perhaps because of a mistake in the gene code. Experts are now looking for a way to cure cancer by modifying faulty DNA to make the p-53 gene work. Lung cancer cells (530x). These cells are from a tumor located in the alveolus (air sac) of a lung.

41 What s Going On Here? Photo of mouse growing a "human ear" - a shape made of cartilage

42 Mixing humans and animals The mythic beast had a lion's body, serpent's tail, and goat's head. Scientists have begun blurring the line between human and animal by producing chimeras a hybrid creature that's part human, part animal. Chinese scientists at the Shanghai Second Medical University in 2003 successfully fused human cells with rabbit eggs. The embryos were reportedly the first human-animal chimeras successfully created. They were allowed to develop for several days in a laboratory dish before the scientists destroyed the embryos to harvest their stem cells.

43 The First Clone! Her name was Dolly.

44 Now cats may have more than nine lives. The company that funded the first successful cloning of a domestic cat, has gone commercial. You can clone your own kitty. Your cost? U.S. $50,000 each.

45 "Cc," the first-ever cloned cat shown here at seven weeks old with Allie, her surrogate mother. The cat was cloned by transplanting DNA from Rainbow, a female three-colored tortoiseshell (or calico) cat into an egg cell whose nucleus had been removed, and then implanting this embryo into Allie, the surrogate mother. "CC's coat color suggests that she is a clone, and a genetic match between CC and the donor mother confirms this," the researchers say.

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47 Scientists at the Oregon Regional Primate Research Center announced the birth of the first genetically engineered primate, named ANDi (for "inserted DNA" spelled backwards), a rhesus monkey whose cells contained the gene that makes jellyfish glow in the dark. The experiment was something of a flop; ANDi does not glow. (Rodents implanted with the gene do.) ANDi

48 The Spider Goat, from the University of Wyoming, was engineered with genes to produce spider silk in its milk. This is important because spider silk is incredibly strong, and has been used in Kevlar vests.

49 Bovine somatotropin BST A hormone composed of protein that is produced by the cows pituitary gland. Helps control the production of milk by assisting the regulation of nutrients into the production of milk or fat.

50 BST Supplementary BST causes the cow to produce less fat and more milk. By splicing genetic material into E. coli bacteria the hormone can be produced at relatively low cost.

51 1982 Humulin is approved for the treatment of diabetes.

52 1. The flounder s antifreeze gene is copied and inserted into a small ring of DNA taken from a bacteria cell. 2. The DNA ring containing the flounder gene is put into a second bacterium. 3. This second bacterium is used to infect the strawberry cell. The flounder s antifreeze gene enters the strawberry s DNA. 4. The new GM strawberry cell is grown into a GM strawberry plant which can be bred many times. Strawberry cell with Antifreeze gene This diagram shows how one type of GM food, a strawberry that resists frost damage is made. The flounder is a fish that live in icy seas. It has a gene that stops it from freezing to death. Strawberries are soft fruits that can easily be damaged by frost. Wonder what they used to make this one blue? A different gene from another organism. Thanks to the new gene, GM strawberries make a protein which helps them resist frost. They don t contain any other fish genes and, and do not taste or smell of fish.

53 Recombinant DNA The ability to combine the DNA of one organism with the DNA of another organism. Recombinant DNA technology was first used in the 1970 s with bacteria.

54 Making Recombinant Bacteria 1. Remove bacterial DNA (plasmid). 2. Cut the Bacterial DNA with restriction enzyme. 3. Cut the DNA from another organism with same restriction enzyme. 4. Combine the cut pieces of DNA together with another enzyme and insert them into bacteria. 5. Reproduce the recombinant bacteria. 6. The foreign genes will be expressed in the bacteria.

55 Basic Steps of Genetic Engineering Step 1: DNA Isolation Isolation of Foreign DNA or - Finding the gene you want to put into another organism. Step 2: Recombinant DNA Insertion of this DNA into Bacterial Plasmid. Step 3: Transformation Insertion of Recombinant plasmid DNA into Bacteria.

56 Step 1 Isolation of Foreign DNA Involves finding the gene you want to sequence. Then cutting it out of the chromosome (DNA) with restriction enzymes that produce sticky ends.

57 Sticky End of EcoR1 EcoRI G AATTC TTAAG S. Stevens

58 HpaI AT HindIII A AGCTT TCGAA PstI CTGCA G ACGT

59 Step 2: Recombinant DNA Formation Involves cutting the vector DNA (the plasmid or other DNA used to deliver the gene chosen) with the same restriction enzyme. Allowing sticky ends to anneal. Bonding the pieces together with Ligase.

60 Recombinant DNA

61 Plasmid Restriction Enzyme Example EcoR1 EcoR1

62 Recombinant DNA Formation

63 Recombinant DNA Formation

64 Recombinant DNA

65 Recombination Insert a foreign gene into a host (Plasmid for example) into the bacterial cell. Goal To produce many copies ( clones) of a particular gene. Reporter gene / Marker Gene tags gene of interest to identify the presence of a gene. Often an antibiotic resistance gene.

66 Distinguishing between transformed and nontransformed cells: Typically involves incorporating an antibiotic resistance gene in the plasmid and then plating the cells on a medium containing that antibiotic. Only the transformed cells are resistant, so only they can grow on the medium.

67 Step 3 Transformation Insert recombinant plasmid into bacteria. Bacteria produced with the recombinant DNA expresses the gene of interest.

68 Vectors The way you get the DNA into the new cell: Plasmids Viruses Particles ( DNA coated bullets) Exogenous DNA

69 Ballistic Gene Transfer - the use of tiny DNAcoated projectiles as carriers. It is important to transport DNA through the walls of intended recipient cells. Projectiles are often known as micro projectiles

70 Plasmids were discovered in the late sixties, and it was quickly realized that they could be used to amplify a gene of interest. A plasmid containing resistance to an antibiotic is used as a vector. The gene of interest is inserted into the vector plasmid and this newly constructed plasmid is then put into E. coli that are sensitive to ampicillin. The bacteria are then spread over a plate that contains ampicillin. As long as you grow the bacteria in ampicillin, it will need the plasmid to survive and it will continually replicate it, along with your gene of interest that has been inserted into the plasmid.

71 Characteristics of a Vector Can replicate independently in the host cell contains an Origin site. Has restriction sites in the vector. 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.

72 Transformation - process of introducing free DNA into bacteria. Competent cell - a cell that is capable of taking up DNA. Electroporation - The use of an electric shock to momentarily open or disrupt cell walls (which allows DNA to enter the cell).

73 Transformation: The purpose of this technique is to introduce a foreign plasmid into a bacteria and to use that bacteria to amplify the plasmid in order to make large quantities of it. This is based on the natural function of a plasmid: to transfer genetic information vital to the survival of the bacteria.

74 3 Types of Transformation:

75 Competent Cells: Since DNA is a very hydrophilic molecule, it won't normally pass through a bacterial cell's membrane. In order to make bacteria take in the plasmid, they must first be made "competent" to take up DNA.

76 This is done by creating small holes in the bacterial cells by suspending them in a solution with a high concentration of calcium. DNA can then be forced into the cells by incubating the cells and the DNA together on ice, placing them briefly at a high temperature (heat shock), and then putting them back on ice. This causes the bacteria to take in the DNA.

77 Competency The procedure to prepare competent cells can sometimes be tricky. Bacteria aren't very stable when they have holes in them, and they die easily. A poorly performed procedure can result in cells that aren't very competent to take up DNA. A well- performed procedure will result in very competent cells. This is typically where the process failed if cells do not transform.

78 S. Stevens Gene (or DNA) Cloning

79 Traditional Cloning is NOT DNA Cloning DNA Cloning (many identical copies of specific DNA molecules) is NOT the same as Organismal Cloning (identical genetic copies of specific individuals).

80 Cloning of Plasmid

81 2 Main purposes of Cloning: 1. It allows for a large number of recombinant molecules to be produced from limited starting materials. 2. Purification limiting the production of extra DNA molecules that do not contain the target gene to be cloned.

82 pglo Gfp Green fluorescent protein

83 Fluorescent In the laboratory, fluorescence is easily achieved by exposing the protein to long range UV light or black" light. The fluorophore absorbs light in the UV-B region (395 nm.. plus a smaller absorbance peak at 470 nm). It emits light (fluoresces) at 509 nm, which is in the green part of the visible spectrum.

84 GFP and Land Mines Neal Stewart at the University of North Carolina is developing plants that can detect land mines. Plants could be ideal biosensors for land mines as seeds would be spread widely and evenly in a suspect field. The gene that can announce the presence of land mines is gfp. The gene will be expressed in the presence of a land mine.

85 GFP and mice

86 Glo fish Fluorescent zebra fish were specially bred to help detect environmental pollutants. By adding a natural fluorescence gene to the fish, scientists are able to quickly and easily determine when waterways are contaminated.