1. 1 Chapter 16 Lecture and Animation Outline Copyright © McGraw-Hill Education. Permission required for reproduction or display. To run the animations you must be in Slideshow View. Use the buttons on the animation to play, pause, and turn audio/text on or off. Please Note: Once you have used any of the animation functions (such as Play or Pause), you must first click on the slide’s background before you can advance to the next slide. See separate PowerPoint slides for all figures and tables pre- inserted into PowerPoint without notes and animations.

2. In 2002, this kitten named “Carbon Copy” was produced by reproductive cloning – the first cloned pet 2

3. Chapter 16 Genetic Technology Gene Cloning Genomics: Techniques for Studying Genomes Biotechnology 3 Chapter Outline:

4. Recombinant DNA technology  Use of laboratory techniques to isolate and manipulate fragments of DNA Recombinant DNA contains DNA from two or more sources Once inside a host cell, recombinant molecules are replicated to produce identical copies or clones 4 Gene Cloning

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6. Gene cloning Creates many copies of a particular gene To make large amounts of a specific gene’s DNA  For DNA sequencing  To probe for a similar gene in another species Or to produce the protein encoded by the gene  Purify protein to study structure and function  Introduce into bacteria or livestock for pharmaceuticals  Introduce into plants and animals to change traits  Use for gene therapy to treat disease 6

7. BIOLOGY PRINCIPLE Biology is an experimental science The technique of gene cloning allows researchers to study genes and gene products in greater detail. 7

8. Gene Cloning – Step 1 Vector DNA is a carrier for the DNA segment to be cloned When a vector is introduced into a living cell, it can replicate, making many copies Common vectors are plasmid or viral vectors Also need the gene of interest from some organism’s chromosomal DNA 8

9. Gene Cloning – Step 2 To insert chromosomal DNA into vector... Cut DNA using restriction enzymes  Also called restriction endonucleases  Made by bacteria as protection against bacteriophages  Cut at specific known restriction sites  Most restriction sites palindromic  May produce sticky ends DNA ligase must be used to permanently link DNA Creates a recombinant vector with gene inserted  Will also get empty recircularized vector without insert 9

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12. Gene Cloning – Step 3 Goal is to have recombinant vector taken up by bacteria  Some will take up a single plasmid  Most cells fail to take up a plasmid at all Vector carries a selectable marker  Adding antibiotics selects for cells expressing antibiotic resistance gene, amp R  Gene amp R codes for beta-lactamase that degrades ampicillin, which normally kills bacteria  Growth on ampicillin plates indicates that bacteria contain plasmid with the selectable marker 12

13. Only the cells with the plasmid grow on plates treated with ampicillin To eliminate recircularized empty vectors, lacZ gene is built into vector  Insertion of cloned DNA disrupts lacZ gene  lacZ codes for beta-galactosidase, which cleaves colorless X-Gal into a blue dye Recircularized plasmids will form blue colonies The desired recombinant vectors will form white colonies 13

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15. DNA library Treatment of chromosomal DNA with restriction enzymes yields tens of thousands of different fragments DNA library – collection of many recombinant vectors each with a fragment of chromosomal DNA Two types of common DNA libraries  Genomic library – inserts derived from chromosomal DNA  cDNA library – uses reverse transcriptase to make complementary DNA (cDNA) from mRNA Lacks introns, so simpler to use 15

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17. Electrophoresis Technique used to separate macromolecules on a gel  Can be used to separate DNA or proteins Can be used to separate molecules based on their charge, size/length, and mass 17

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19. Polymerase chain reaction (PCR) Allows us to amplify DNA (make many copies)  No vectors or host cells needed Ingredients:  Primers that match sequences at each end of the DNA fragment  dNTPs (deoxynucleotide triphosphates)  Taq polymerase – a heat-stable form of DNA polymerase called DNA run through repeated cycles of denaturation, annealing and synthesis  Thermocycler machine automates this process After 30 cycles of amplification, a DNA sample will increase 2 30 -fold 19

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24. 24 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.

25. Genomics – the molecular analysis of the entire genome (all the DNA) of a species Two phases  Mapping of genome  Functional genomics How do specific DNA sequences impact structure and function of cells and the organism? Expression analysis – which genes turn on or off in particular cells 25 Genomics: Techniques for Studying Genomes

26. DNA sequencing Determines base sequence of DNA Dideoxy chain-termination method  Dideoxynucleoside triphosphates (ddNTPs) are missing the 3’–OH group and cause chain to terminate  4 tubes with copies of the single stranded DNA, each with a different labelled nucleotide  DNA polymerase will make complementary strand until ddNTP inserted and chain terminates  After electrophoresis, DNA sequence can be read by determining which base is at the end of the DNA strand Procedure has been automated using fluorescently labeled ddNTPs all in one tube 26

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28. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 3′ Recombinant vector Annealing site 5′ Primer Many copies of the recombinant vector, primer, dNTPs, fluorescently labeled dideoxynucleotides (ddNTPs), and DNA polymerase are mixed together. Incubate to allow the synthesis of DNA. CACCGTAAGGACTddG CACCGTAAGGACddT CACCGTAAGGAddC CACCGTAAGGddA CACCGTAAGddG CACCGTAAddG CACCGTAddA CACCGTddA CACCGddT CACCddG CACddC CAddC CddA ddC Separate newly made strands by gel electrophoresis. 9 8 1 2 3 4 5 6 7 10 11 12 13 14 G T G G C A T T C C T G A C Sequence to be analyzed (target DNA) Synthesis of short DNA strands until a ddNTP is added. G T C A G G A A T G C C A C Sequence deduced from gel (a) Dideoxy DNA sequencing method Fluorescence detector CACCGTAAGGACTG (b) Output from automated sequencing Longest DNA strand Laser beam

29. 29 Please note that due to differing operating systems, some animations will not appear until the presentation is viewed in Presentation Mode (Slide Show view). You may see blank slides in the “Normal” or “Slide Sorter” views. All animations will appear after viewing in Presentation Mode and playing each animation. Most animations will require the latest version of the Flash Player, which is available at http://get.adobe.com/flashplayer.

30. A microarray can identify which genes are transcribed by a cell DNA microarray or gene chip Monitors expression of thousands of genes simultaneously Short sequences of known genes attached to spots on slide Goal is to find out which genes are transcribed into mRNA in a particular sample of cells  mRNA from those cells is used to make fluorescently labeled cDNA cDNAs that are complementary to the DNAs in the microarray will hybridize High fluorescence intensity in a spot indicates high levels of cDNA in the sample

33. Technologies that involve the use of living organisms to benefit humans  12,000 years ago – domestication of livestock  Now – molecular genetics Important medicines are now produced by recombinant organisms  Tissue plasminogen activator – for blood clots  Factor VIII – for hemophilia  Insulin – for diabetes 33 Biotechnology

34. Insulin In 1982, the U.S. FDA approved sale of human insulin made by recombinant bacteria Prior to 1982, insulin was isolated from cattle  Some people developed allergies and had to use cadaver insulin Insulin composed of two polypeptides – A and B  A and B coding sequence inserted into E.coli  Fusion proteins extracted and β-galactosidase removed  Purified A and B chain mixed to form functional protein 34

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36. BIOLOGY PRINCIPLE Biology affects our society This method of making insulin, which was developed by researchers, is now used to make insulin for most people with insulin-dependent diabetes. 36 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

37. Gene replacement and knockout Used to understand gene function and human disease A transgenic organism carries genes introduced using molecular techniques  Also called Genetically Modified Organism (GMO) Gene replacement – cloned gene recombines with normal gene on a chromosome  Only 1 of 2 copies replaced, creating heterozygote  Heterozygotes can be crossed to yield homozygotes 37

38. Gene knockout – if cloned gene is a mutation that inactivates function, homozygote will not have gene function Very useful for studying human disease  Used to create models to study disease  Used to test effect of therapies 38

39. Molecular pharming Production of medically important proteins in livestock mammary glands Certain proteins are more likely to function when expressed in mammals  Post-translational modification  Degraded or improperly folded in bacteria  High yield in cows 39

40. Strategy: Clone gene next to the promoter of a gene specifically expressed in mammary cells Vector injected into oocyte, fertilized and implanted Protein is purified out of milk 40

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42. Transgenic plants Somewhat easier because plant cells are totipotent Cloned genes can be introduced into somatic tissue and entire plant regenerated using hormones Agrobacterium tumefaciens naturally infects plant cells and causes tumors  Contains Ti plasmid that integrates into host chromosome  Codes for plant growth hormones that form crown gall tumor 42

43. Transgenic plants Ti plasmid modified to introduce cloned genes  Kan R used as a selectable marker for kanamycin resistance  Contain convenient restriction sites Transformed cells plated on media with kanamycin (to kill nontransformed cells) and carbenicillin (to kill Agrobacterium) 43

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45. Successful example of the use of transgenic plants has involved the introduction of genes from Bacillus thuringiensis (Bt) Bacterium produces toxins that kill certain caterpillars and beetles and has been widely used as an insecticide for several decades Bt varieties of plants produce the toxins themselves – therefore are resistant to many caterpillars and beetles 45

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47. BIOLOGY PRINCIPLE Biology affects our society The field of agriculture has been greatly influenced by the development of genetically modified organisms (GMOs) such as Bt corn. 47 Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display.

48. Cloning in mammals Identical twins are genetic clones from a single fertilized egg that split early in development Plants can be cloned from somatic cells For decades it was believed that mammalian somatic cells could not be used for cloning 1996, Dolly was the first cloned lamb 48

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50. Mammary cells from adult sheep removed Fused diploid mammary cell with enucleated sheep oocyte Zygote implanted Dolly and donor were almost genetically identical  Same set of genes  Minor differences due to differences in mitochondrial DNA and maternal effect genes Cloning achieved now in several mammal species 50

51. Stem cells Have potential to treat a variety of human disorders Stem cells are undifferentiated cells that can divide and create the bodies of plants and animals  They have the capacity to divide  Can differentiate into specialized cell types Embryonic stem cells (ES cells) and embryonic germ cells (EG cells) are pluripotent  They can differentiate into many different cell types  Thus potential for many different diseases Stem cells already in use in bone marrow transplants 51

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54. Blaese and colleagues performed the first gene therapy to treat ADA deficiency Gene therapy puts cloned genes into living cells to cure disease Over 4,000 diseases involve mutations in single genes Adenosine deaminase deficiency prevents proper metabolism of nucleosides Deoxyadenosine accumulates and results in destruction of B and T cells  Leads to severe combined immunodeficiency syndrome (SCID)  Fatal at an early age (1 or 2 years old) FEATURE INVESTIGATION

55. Three treatment approaches:  Bone marrow transplant  Purified ADA enzyme  Gene therapy Gene therapy  September 14, 1990  Removed lymphocytes from girl  Treated with retroviral vector containing ADA gene  Returned cells to her bloodstream Results suggest that this first gene therapy trial may offer benefit, but patients were also treated with ADA – so could not know for sure FEATURE INVESTIGATION

56. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. 1 2 4 Retrovirus 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. KEY MATERIALS A retrovirus with the normal ADA gene. Remove ADA-deficient lymphocytes from the patient with severe combined immunodeficiency disease (SCID). Culture the cells in a laboratory. Lymphocyte Infuse the ADA-gene-corrected lymphocytes back into the SCID patient. Lymphocytes Lymphocyte X Mutant ADA gene Normal ADA gene Retroviral inserted DNA X Experimental levelConceptual level 3 Infect the lymphocytes with a retrovirus that contains the normal ADA gene. As described in Chapter 18, retroviruses insert their DNA into the host cell chromosome as part of their reproductive cycle. Retrovirus with ADA gene FEATURE INVESTIGATION

57. Copyright © The McGraw-Hill Companies, Inc. Permission required for reproduction or display. ADA function from gene therapy (nmoles of deaminated deoxyadenosine/10 8 cells) 6 7 03657301,0951,460 Protocol day 20 5 0 10 15 5 CONCLUSION The introduction of a cloned ADA gene into lymphocytes via gene therapy resulted in higher ADA function, even after 4 years. SOURCE Blaese, R. Michael et al. 1995. T lymphocyte- directed gene therapy for ADA-SCID: Initial trial results after 4 years. Science 270:475–480. THE DATA Transfusions stopped FEATURE INVESTIGATION