1. Chapter 19 *Lecture Outline Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. *See separate FlexArt PowerPoint slides for all figures and tables pre-inserted into PowerPoint without notes.

2. INTRODUCTION Biotechnology is broadly defined as technologies that involve the use of living organisms, or their products, to benefit humans It is not a new topic –It began about 12,000 years ago when humans began to domesticate animals and plants for the production of food Since the 1970s, molecular genetics has provided new, improved ways to make use of organisms to benefit humans –Genetically modified organisms (GMOs) have received genetic material via recombinant DNA technology –An organism that has integrated recombinant DNA into its genome is called transgenic 19-2 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

3. 19.1 USES OF MICROORGANISMS IN BIOTECHNOLGY Microorganisms are used to benefit humans in various ways –Refer to Table 19.1 Molecular genetic tools are very important in influencing and improving our use of microorganisms Overall, the use of recombinant microorganisms is an area of great research interest and potential –However, there are problems such as safety concerns and negative public perception Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-3

4. 19-4

5. Many Important Medicines Are Produced by Recombinant Microorganisms 19-5

6. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Insulin regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells It is produced by the  cells of the pancreas Persons with insulin-dependent diabetes have a defect in their  cells Therefore, they cannot synthesize enough insulin Sources of insulin included Cows Human cadavers! But now, patients can use insulin made by recombinant bacteria Refer to Figure 19.1 19-6 Insulin

7. P O P O lacZ amp R  galactosidase  galactosidase Met CNBr cleaves the peptide bond after methionine. Met B chain Insulin B chain Treat with CNBr.Purify β-galactosidase-. insulin fusion proteins. Culture cells.Transform into E. coli.Purify A and B chains. Active insulin Disulfide bond Refolding and disulfide bond formation Met A chain Insulin A chain Met Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Figure 19.1 The use of bacteria to make human insulin 19-7 Insulin is a hormone composed of two polypeptide chains, called the A and B chains

8. Bacterial Species Can Be Used as Biological Control Agents Biological control refers to the use of microorganisms or their products to alleviate plant problems –Disease or damage from environmental conditions Biological control agents can prevent disease in one of two main ways: –1. Nonpathogens are used to compete effectively against pathogens for nutrients or space –2. Microorganism may produce toxins that inhibit other microorganisms or insects, but not the plant Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-8

9. Biological control can also involve the use of microorganisms in the field –A successful example is the use of Agrobacterium radiobacter to prevent crown gall disease caused by Agrobacterium tumefaciens –A. radiobacter produces agrocin 84, an antibiotic that kills A. tumefaciens A. radiobacter contains genes that confer resistance to agrocin 84 –The genes responsible for agrocin 84 synthesis and resistance are on a plasmid Unfortunately, this plasmid can be transferred via conjugation –Researchers have found A. radiobacter strains with plasmids that have lost the ability to be transferred These conjugation-deficient strains are now used commercially worldwide to prevent crown gall disease Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-9

10. Biological control can also involve the use of microorganisms in the field –Another successful example is the use of Bacillus thuringiensis toxins. These ‘Bt’ toxins are lethal to many caterpillars and beetles Generally harmless to plants and humans Bt toxins are sold in powder form –Can be used as foliage spray –Toxins paralyze insect’s digestive tract Will discuss later how Bt is used to produce transgenic plants Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-10

11. The Release of Recombinant Microorganisms into the Environment A recombinant microorganism is one whose DNA is altered in vitro and then reintroduced back into it –Mutations and acquisition of naturally-occurring plasmids do not create recombinant strains!!! This is an important distinction from the perspective of governmental regulation and public perception –Each year new strains of nonrecombinant microorganisms are analyzed in field tests for biological control of plants –However, the release of recombinant microorganisms is controversial Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-11

12. As an example, let’s consider the first field test of a recombinant bacterium –It was carried out by Steven Lindow and colleagues –It involved the use of genetically-engineered Pseudomonas syringae to control frost damage Ice + strains produce proteins that promote ice nucleation Ice – strains do not make ice-nucleation proteins When applied to the surface of plants, an Ice – strain can compete with, and thereby reduce, the proliferation of Ice + bacteria –Lindow sought approval for field tests of an Ice – recombinant strain in Tulelake, California These tests were delayed several years by a lawsuit When the tests were finally approved in 1987, vandals struck! Nevertheless, the results of the field experiment did show that Ice – bacteria did protect potato plants from frost damage (Figure 19.2) However, the product was never commercialized Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-12

13. Microorganisms Can Reduce Environmental Pollutants The term bioremediation refers to the use of microorganisms to reduce environmental pollutants –During bioremediation, enzymes produced by a microorganism transform the structure of the toxic pollutant –This event is called biotransformation In many cases, biotransformation results in biodegradation –The toxic pollutant is degraded into nontoxic metabolites Biotransformation without biodegradation can occur –In these cases, the pollutant is rendered less toxic by Oxidation or reduction reactions Polymerization reactions Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-13

14. Since the early 1900s, microorganisms have been used in the treatment and degradation of sewage –More recently, the field of bioremediation has expanded into the treatment of hazardous and refractory wastes These wastes are associated with chemical and industrial activity –These pollutants include petroleum hydrocarbons, pesticides, herbicides, organic solvents, etc. In 1980, the U.S. Supreme Court ruled that a live, recombinant microorganism is patentable –As a “manufacture or composition of matter” –The first patented organism was an “oil-eating” bacterium But alas, it has not been a commercial success –It can metabolize only a few of the 3,000 or so toxic compounds in crude oil Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-14

15. 19.2 GENETICALLY MODIFIED ANIMALS The production of transgenic animals is a relatively new, exciting area of biotechnology –It holds great promise for innovations in biotechnology Of course, this is predicated on public acceptance! Figure 19.3 shows a transgenic mouse that expresses the human growth hormone gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-15

16. Figure 19.3 19-16

17. Gene Addition versus Gene Replacement Cloned genes can be introduced into plant and animal cells –However, the gene will not be inherited stably if it does not become integrated into the host cell’s genome This integration occurs by recombination The introduction of a cloned gene into a cell can lead to one of two outcomes –Gene replacement (can lead to gene knockout if a defective copy replaces a good copy.) –Gene addition –Refer to Figure 19.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-17

18. + Cloned gene Homologous recombination Normal gene Cloned gene (a) Gene replacement + Cloned gene Nonhomologous recombination Normal gene Cloned gene Normal gene (b) Gene addition Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19-18 Figure 19.4 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display If the cloned gene was rendered inactive by mutation => gene knockout

19. 19-19 Figure 19.5 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Genes can be introduced from different species. Here, different fluorescent proteins from jellyfish are expressed in zebrafish.

20. Production of Mice That Contain Gene Replacements In bacteria and yeast, gene replacement is the common outcome –These have relatively small genomes, so homologous recombination occurs at a relatively high rate In complex eukaryotes, gene addition is the norm –These have very large genomes, so homologous recombination is rare Only 0.1% of the time Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-20

21. To produce mice with gene replacements, molecular biologists have resorted to trickery –Cells in which homologous recombination has occurred are preferentially selected This approach is shown in Figure 19.6 –The cloned gene is altered using two selectable markers A neomycin-resistant gene (Neo R ) is inserted into the center of the coding sequence of the target gene A thymidine-kinase gene (TK) is inserted adjacent (not within) the target gene –TK renders cells sensitive to killing by a drug called gancyclovir –The modified target gene is then introduced into mouse embryonic cells which can be grown in the lab Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-21

22. Neo R TK Gene of interest Normal gene Neo R Chromosomal DNA TK Dies Neo R Survives This cloned DNA is then introduced into embryonic stem cells. In this case, the cells were derived from a mouse with dark fur color. The cells are grown in the presence of neomycin and gancyclovir. Only those cells that contain the Neo R gene but are lacking the TK gene will survive. The gene of interest has been cloned. A neomycin resistance gene is inserted into the center of this gene, and a thymidine kinase gene is inserted next to the gene. –Same gene as normal, chromosomal gene except it has Neo R inserted into it. Nonhomologous recombination Embryonic stemc ells Homologous recombination 19-22 Figure 19.6 Sensitive to gancyclovir Resistant to both drugs Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

23. Blastocyst Chimeric offspring Surviving cells are injected into embryonic blastocysts derived from a mouse with white coat color. The injected blastocysts are reimplanted into the uterus of a female mouse. © Alan Handyside, Wellcome Images Neo R Survives 19-23 Figure 19.6 A chimera is an organism that contains cells from two different individuals Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Following birth, chimeric mice are identified as those that contain a coat with both dark and white fur. The appropriate crosses are made in order to produce mice that have two copies of the target gene. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

24. 19-24 Gene Knockouts help scientists understand human diseases Gene knockouts may reveal the function of the gene Sometimes there is no obvious phenotype –Single gene may only make small contribution to overall phenotype –Another gene with similar function may compensate This is called gene redundancy Knocking out mouse genes may teach us about human disease –Mice and humans share many genes Seeing characteristics of knockout mice may lead to treatments of human diseases. –Mouse models have been useful in understanding cancer, obesity, heart disease, diabetes and many inherited diseases. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display

25. Gene Knockin A Gene Knockin is a gene addition in which a gene of interest has been added to a particular site (usually noncritical) in a genome. 19-25 Figure 19.7 The gene of interest is cloned with flanking pieces of DNA from a noncritical site in the mouse genome. The cloned DNA is introduced into an embryonic stem cell. The gene of interest inserts into noncritical site by homologous recombination. Nucleus of a mouse embryonic stem cell Mouse chromosome Gene of interest Noncritical site Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

26. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Transgenic Livestock Transgenic species of livestock are being developed –Includes fish, sheep, pigs, goats and cattle as well as others May include production of medicines in the milk of these animals –Sometimes called molecular pharming –See Figure 19.8 and Table 19.3 19-26

27. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-27 Figure 19.8 Table 19.3 Purify the hormone from the milk. Obtain milk from female transgenic sheep. The milk contains a human hormone. Inject this DNA into a sheep oocyte. The plasmid DNA will integrate into the chromosomal DNA, resulting in the addition of the human hormone gene into the sheep's genome. Implant the fertilized oocyte into a female sheep, which then gives birth to a transgenic sheep offspring. Using recombinant DNA technology (described in Chapter 18), clone a human hormone gene next to a sheep β-lactoglobulin promoter. This promoter is functional only in mammary cells so that the protein product is secreted into the milk. Human hormone gene Plasmid vector Transgenic sheep β-lactoglobulin promoter Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

28. 19.3 REPRODUCTIVE CLONING Reproductive cloning refers to methods that produce two or more genetically identical individuals –Identical twins are genetic clones from one fertilized egg Cloning is an easier undertaking in plants –Plants can be cloned from somatic cells For several decades scientists believed that mammalian somatic cells were unsuitable for cloning –But in 1997, Ian Wilmut and his colleagues at the Roslin Institute created Dolly! Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-28

29. Donor sheep's mammary cell is extracted and grown in a tissue culture flask. Another sheep's unfertilized egg is extracted, and the nucleus is removed. The donor nucleus from the mammary cell and the maternal proteins within the enucleated egg initiate development of the egg into an embryo. Mammary cell Donor sheep Unfertilized egg Nucleus Mammary cell The cells are fused together with electrical pulses. Egg with nucleus removed 19-29 Figure 19.9 Protocol for the successful cloning of sheep Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

30. 19-30 Figure 19.9 Protocol for the successful cloning of sheep Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The embryo is transferred into a surrogate ewe. Allow pregnancy to proceed. Surrogate ewe A lamb genetically identical to the donor sheep is then born. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

31. Evidence suggested that Dolly may have been “genetically older” than her actual age would have indicated –At 3 years old, the length of the telomeres in her somatic cells were consistent with a sheep that is 9 or 10 years old The sheep that donated the somatic cell that produced Dolly was 6 years old –Thus, Dolly’s shorter telomeres were likely a result of the shortening of telomeres in the donor sheep In 2003, 6-year old Dolly was euthanized after an examination showed progressive lung disease –Her death raised concerns that the techniques used to produce Dolly could have caused premature aging –Microarray studies in cloned mice showed as much as 4% of genes were not expressed normally Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-31

32. In recent years, cloning from somatic cells has been achieved in several mammalian species –Sheep, cows, mice, goats and pigs –Unlike the case with Dolly, telomeres in cloned mice and cattle appear to be the correct length! –However, other studies have shown other genetic flaws With regard to livestock, farmers can use somatic cells from their best individuals to create genetically homogeneous herds –This may be advantageous with regard to agricultural yield –However, such a herd may be more susceptible to rare diseases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-32

33. People have become greatly concerned with the possibility of human cloning –To some, it is morally wrong and threatens the basic fabric of parenthood and family –To others, it offers a new avenue of reproduction For infertile couples who might want a genetically related child In the public sector, the sentiment toward human cloning has been generally negative –Indeed, many countries have issued an all-out ban –While others permit limited research in the area In the future, our society will have to wrestle with the legal and ethical aspects of cloning Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-33

34. Stem Cells Stem cells supply the cells that construct our bodies from a fertilized egg –In the adult, stem cells also replenish damaged cells Stem cells have two common characteristics –1. They have the capacity to divide –2. They have the capacity to differentiate into one or more specialized cell types totipotent cells, like fertilized eggs can give rise to all cell types pluripotent cells can differentiate into almost every cell, but can’t give rise to an entire, intact individual multipotent cells can differentiate into several cell types unipotent cells can only differentiate into one cell type –Refer to Figures 19.11 and 19.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-34

35. 19-35 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Figure 19.11 When a stem cell divides, one may remain undifferentiated, while the other can differentiate into a specialized cell type –Thus the population of stem cells remains constant Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Cellular division Stem cell Red blood cell + + Stem cell Differentiation

36. Blastocyst Inner cell mass Embryonic stem cells (ES cells) (pluripotent) Fertilized egg (totipotent) Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19-36 In mammals, stem cells are commonly categorized based on their developmental stage and their ability to differentiate Figure 19.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Can produce all cell types in an adult organism Can give rise to an entire organism Can produce almost all cell types in an adult organism However, a single cell has lost the ability to produce an entire organism Found in the early mammalian embryo

37. Fetus Embryonic germ cells (EG cells) (pluripotent) Many types of adult stem cells (multipotent or unipotent) 19-37 Figure 19.12 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Certain types of human cancers called teratocarcinomas arise from pluripotent cells These cells are called embryonic carcinoma cells (EC cells) Can only differentiate into a single cell type For example, primordial germ cells in the testis  sperm, only Can differentiate into several cells types For example, hematopoietic stem cells (HSC cells) of the bone marrow Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

38. 19-38 Figure 19.13 Fates of hematopoietic stem cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display A Multipotent stem cell can give rise to many different cell types Hematopoietic stem cell Hematopoietic stem cell + or Myeloid progenitor Red blood cell Basophil Platelets Megakaryocyte Osteoclast Monocyte Macrophage Eosinophil Neutrophil Dendritic cell Lymphoid progenitor Hematopoietic progenitor Cell division T cell B cell Natural killer cell Dendritic cell Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

39. Stem Cells Interest in stem cells centers around two main areas –1. They may help us understand the basic genetic mechanisms that underlie the process of development –2. They offer the potential to treat human diseases or injuries that cause cell and tissue damage This application has already become a reality –For example, bone marrow transplants are used to treat patients with certain forms of cancer –As shown in Table 19.4, embryonic stem cells could potentially be used to treat a wide variety of diseases Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-39

40. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-40

41. Stem Cells Adult stem cells are rare –1 cell in 10,000 in the bone marrow is a stem cell Embryonic Stem cells (ES) and Embryonic Germ cells (EG) can be grown in the laboratory –Are easily identified –Provide greatest potential for transplantation therapy –Most ES cells are derived from unused embryos from in vitro fertilization –Most EG cells are derived from aborted fetuses –This creates an ethical dilemma in using these cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-41

42. 19.4 Genetically Modified Plants Selective breeding has been used for centuries –This has produced plants with desirable characteristics larger, disease resistance, high-quality food Genetically engineered crops have been used since mid-1990s –In 2009, roughly 25% of all crops were transgenic –More than 100 million hectares planted with transgenics Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-42

43. A. Tumefaciens Can Be Used to Make Transgenic Plants The production of transgenic plants is somewhat easier than transgenic animals –Certain plant cells are totipotent An entire organism can be regenerated from a somatic cell Agrobacterium tumefaciens is a bacterium that naturally infects plants causing crown gall tumors –Ti plasmid (Tumor-inducing) carried by bacterium –Refer to Figure 19.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-43

44. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19-44 Tumor-inducing plasmid Transferred DNA Figure 19.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display During infection, the T DNA within the Ti plasmid is transferred to the plant cell. The T DNA becomes integrated into the plant cell's DNA. Genes within the T DNA promote uncontrolled plant cell growth. Plant chromosome T DNA A. tumefaciens Ti plasmid Plant cell Wound site Agrobacterium tumefaciens is found within the soil. A wound on the plant enables the bacterium to infect the plant cells. T DNA

45. 19-45 Figure 19.14 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display T DNA The growth of the recombinant plant cells produces a crown gall tumor. (a) The production of a crown gall tumor by A. tumefaciens infection Crown gall tumor T DNA Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

46. The A. tumefaciens T DNA can be used as a vector to introduce cloned genes into plants First, the Ti plasmid needs to be modified –The genes that cause tumors are deleted –Selectable marker genes are inserted into the T DNA Kan R is commonly used –Unique restriction sites are added for the convenient insertion of any gene Figure 19.15 shows the general strategy for producing transgenic plants via T DNA-mediated gene transfer Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-46

47. 19-47 Figure 19.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Gene of interest is inserted into the T DNA of the T-DNA vector. Gene of interest T-DNA vector Kan R (confers kanamycin resistance) T DNA Site where restriction enzyme cuts Recombinant T-DNA vector The recombinant T-DNA vector is transformed into A. tumefaciens. Plant cells are exposed to A. tumefaciens. The T DNA is transferred and incorporated into the plant cell chromosome. Carbenicillin and kanamycin are then added to kill A. tumefaciens cells and plant cells that have not taken up T DNA, respectively. Recombinant T-DNA vector A. tumefaciens

48. Plant cell The plant cells are transferred to a medium containing growth hormones to regenerate an entire plant. Inserted T DNA carrying new gene Plant with cloned gene 19-48 Figure 19.15 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display T DNA integrates into the genome Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

49. A. tumefaciens does not infect all plant species –Fortunately, other methods are available for introducing genes into plant cells –Biolistic gene transfer (i.e., biological ballistics) The second most common way to produce transgenic plants A “DNA gun” is used to shoot DNA-coated microprojectiles into the cells –Microinjection Microscopic-sized needles are used to inject DNA into the cells –Electroporation An electric current is used to create transient pores in the plasma membrane through which DNA can enter Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-49

50. Transgenic Plants Transgenic plants can be given characteristics that are agriculturally useful –For example, the Monsanto Company has produced plants highly tolerant of glyphosate The active ingredient in the herbicide Roundup TM –Compared to nontransgenics, these plants grow quite well in the presence of glyphosate-containing herbicides –Plants have been made more disease resistant Table 19.5 presents some of the various traits that have been modified in plants Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-50

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52. Transgenic plants have been approved for human consumption –The first example was the Flavr Savr tomato (Figure 19.18) This transgenic plant has been given the gene that encodes an antisense RNA –Complementary to the mRNA for polygalacturonase (an enzyme involved in fruit ripening) The antisense RNA binds to the mRNA and prevents translation In addition, double-stranded RNA is targeted for degradation –RNAi-mediated silencing (refer to Chapter 15) –The practical advantage of the Flavr Savr tomato is improved shelf-life It does not spoil (overripen) as quickly as traditional tomatoes Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-52

53. Transgenic plants can produce their own insecticide –Express genes from the naturally occurring bacterium Bacillus thuringiensis (Bt) –These genes express toxins that are lethal to many caterpillars and beetles that feed on trees, shrubs and fruit –Bt corn and cotton are very successful (see Figure 19.18) There are concerns about the use of Bt –may kill pollinators of native species –may cause proliferation of resistant insects Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-53

54. Production of Bt crops is rising Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-54 Figure 19.18 (a) A field of Bt corn (b) Bt corn and Bt cotton usage since 1996 Percent of acres 100 0 20 40 60 80 Year Bt cotton Bt corn 199697989920000102030405060708 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. © Bill Barksdale/AGStockUSA

55. 19.5 HUMAN GENE THERAPY Gene therapy is the introduction of cloned genes into living cells in an attempt to cure disease Research efforts in gene therapy are aimed at –Alleviating inherited diseases –Treating diseases such as cancer and heart disease –Combating infectious diseases such as AIDS Human gene therapy is still at an early stage of development –Success has been limited –Nevertheless, some of the initial results are promising and future prospects abound –Refer to Table 19.6 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-55

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57. Gene Therapy Involves the Introduction of Cloned Genes into Human Cells Two transfer methods are used –1. Nonviral approach Refer to Figure 19.19a Liposome technique most common –2. Viral approach Refer to Figure 19.19b Most common are retroviruses, adenoviruses and parvoviruses Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-57

58. 19-58 Figure 19.19 Carries a positive charge (cationic) Virus is genetically altered so that it cannot proliferate after entry into host cells Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Retrovirus- RNA genome contains gene of interest. Liposome DNA carrying the gene of interest Target cell The liposome is degraded within the endosome and the DNA is released into the cytosol. DNA-liposome complex is taken into the target cell by endocytosis. The DNA is imported into the cell nucleus. Retrovirus is taken into the target cell via endocytosis. The RNA is reverse transcribed into DNA, which travels into the nucleus. RNA genome The viral coat is disassembled in the endosome, and two copies of the RNA genome are released into the cytosol.

59. 19-59 Figure 19.19 Does not elicit immune response Elicits immune responseLow efficiency High efficiency ADVANTAGE DISADVANTAGE Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display (a) Nonviral approach(b) Viral approach Endosome Reverse transcriptase Integrated gene Integrated gene By recombination, the DNA carrying the gene of interest is integrated into a chromosome of the target cell. By recombination, the viral DNA, carrying the gene of interest, is integrated into a chromosome of the target cell. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

60. Experiment 19A: The First Human Gene Therapy Adenosine deaminase (ADA) is an enzyme involved in purine metabolism –If both copies of the gene are defective, deoxyadenosine will accumulate within the cells of the individual –Deoxyadenosine is particularly toxic to B and T cells –The destruction of these cells leads to a disease termed severe combined immunodeficiency (SCID) –If left untreated, SCID is typically fatal at an early age Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-60

61. Three approaches can be used to treat adenosine deaminase (ADA) deficiency –1. A bone marrow transplant from a compatible donor –2. Purified ADA coupled to polyethylene glycol (PEG) –3. Gene therapy On September 14, 1990 the first human gene therapy was approved for a girl with ADA deficiency –Prior to the clinical trial, the normal ADA gene had been cloned into a retrovirus that can infect lymphocytes –The general aim of the therapy was to remove lymphocytes from a patient with SCID, culture the cells in the laboratory, introduce the normal ADA gene into the cells and reintroduce the cells into the patient Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-61

62. Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-62 The Hypothesis Infecting lymphocytes with a retrovirus containing the normal ADA gene will correct the inherited deficiency of the mutant ADA gene in patients with ADA deficiency. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display Testing the Hypothesis Refer to Figure 19.20

63. 19-63 Figure 19.20 This is called an ex vivo approach The genetic manipulations occur outside the body, yet the products are reintroduced into the body Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. Experimental level Conceptual level 2. Culture the cells in a laboratory. 1. Remove ADA-deficient lymphocytes from the patient with severe combined immunodeficiency disease (SCID). Lymphocytes Lymphocyte Mutant ADA gene X

64. 19-64 Figure 19.20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display Retroviral inserted DNA 3. Infect the cells with a retrovirus that contains the normal ADA gene. Retroviruses insert their DNA into the host cell chromosome as part of their reproductive cycle. 4. Infuse the ADA-gene-corrected lymphocytes back into the SCID patient. Retrovirus with ADA gene LymphocyteRetrovirus Normal ADA gene X Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

65. ADA function from gene therapy (nmoles of deaminated deoxyadenosine/10 8 cells) 20 0 5 10 15 Protocol day 036514607301095 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 19-65 Figure 19.20 Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display The Data DNA was transferred into living cells

66. Interpreting the Data Results of treatment were not conclusive –ADA gene was expressed in only a small percentage of cells –Drug treatment still needed In another test of  c cytokine receptor gene therapy, patients developed leukemia –caused by integration of the retroviral vector used to treat the disease next to a particular gene Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-66

67. Aerosol Sprays May Be Used to Treat Cystic Fibrosis Cystic fibrosis (CF) is one of the most common recessive inherited disorders –Affects about 1 of 3,000 babies of northern European descent –It is caused by a defect in a gene that encodes an ion transport protein –This leads to an abnormality in salt and water balance –This, in turn, leads to accumulation of mucus in the lungs –The result is chronic lung infections which prove fatal Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-67

68. CF has been the subject of much gene therapy research –To implement CF gene therapy, the normal CF gene must be delivered to lung cells The ex vivo approach used in ADA gene therapy is not possible CF gene therapy has involved the use of an aerosol spray –In one approach, the normal CF gene is delivered in an adenovirus –In another, the gene is delivered via liposomes –When inhaled by the patient via an aerosol spray, the lung epithelial cells take up liposomes and adenoviruses –Still at an early stage of development –Eventually may become an effective method of treatment Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display 19-68