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Genetic engineering

  1. GENETIC ENGINEERING By, Dr. Priyanka Sharma II year MDS Department of Public Health Dentistry JSS Dental College & Hospital 1 1
  3. CONTENTS 3 3
  4. INTRODUCTION Genetic engineering is a part of biotechnology. Biotechnology is the use of living systems and organisms to develop or make useful products, or "any technological application that uses biological systems, living organisms or derivatives thereof, to make or modify products or processes for specific use" (UN Convention on Biological Diversity, Art. 2). 4 4
  5. INTRODUCTION continuation..  Biotechnology is a huge topic.  Its hard to define its exact boundaries.  Some European scientists divide the field into : 1) Red biotechnology 2) Green biotechnology  Some divides it into : 1) White 2) Blue  Biotechnology falls under many umbrellas which is basically considered as life science. Book : Biotechnology & Genetic engineering (Kathy wilson peacock) 5 2010,Edi:1 : Page No. 4 (Chapter 1) 5
  6. Biology & Zoology Cell biology Microbiology Molecular Biology Physiology, Ecology, Embryology Genetics, Population genetics, Epigenetics Proteonomics & Bioinformatics Book : Biotechnology & Genetic engineering 6 6
  7. 7 INTRODUCTION continuation.. • Genetics – science of genes, heredity and variation in living organisms. • Genetics deals with the molecular structure and function of genes, and gene behavior in context of a cell or organism (e.g. dominance and epigenetics ). • Patterns of inheritance from parent to offspring, and gene distribution, variation and change in populations = Population genetics. 7 Book : Genetics and the Organism: Introduction
  8. 8 INTRODUCTION continuation.. Essence Of Genetics • Chromosome • Packaged and organized chromatin, a complex of macromolecules found in cells, consisting of DNA, protein and RNA. Essence Of Genetics • DNA • A molecule that encodes the genetic instructions used in the development and functioning of all known living organisms and many viruses. Essence Of Genetics • Genetic Variation • Permanent change in the chemical structure of genes brought about by mutation, important in providing genetic material for natural selection. Essence Of Genetics • Heredity • The study of heredity in biology is called genetics, which includes the field of epigenetics. 8 Book : Genetics and the Organism: Introduction
  9. 9 9 U S National Library of Medicine
  10. 10 10 A form – 11 bp/ turn B form- 10 bp/ turn Z form- 12 bp/ turn From Lippincotts - Illustrated Biochemistry
  11. 11 11 Deletion Inversion Duplication Insertion Translocation
  12. 12
  13. INTRODUCTION continuation.. Various Branches of Genetics Behavioural genetics Classical genetics Developmental genetics Conservation genetics Ecological genetics Evolutionary genetics Genetic engineering & Metagenesis 13 13 Book : Genetics and the Organism: Introduction
  14. CONTENTS 14 14
  15. BASICS OF GENETIC ENGINEERING • Different terms used for genetic engineering : 1) Gene manipulation 2) Gene cloning 3) Recombinant DNA technology 4) Genetic modification 5) New genetics An Introduction to Genetic Engineering (Desmond S. T. Nicholl) Edi :3rd 2008 15 Chapter 2 . Page 3 15
  16.  Direct manipulation of an organism's genome using biotechnology . First isolating and copying the genetic material of interest using molecular cloning methods Generate a DNA sequence New DNA inserted in the host genome BASICS OF GENETIC ENGINEERING CONTINUATION.. An Introduction to Genetic Engineering (Desmond S. T. Nicholl) Edi :3rd 2008 Chapter 2. 16
  17. 17 Miller et al(2000). An Introduction to Genetic Analysis (7th ed.). 17
  19. Genetic inheritance was first discovered by Gregor Mendel in 1865 following experiments crossing peas. • Although largely ignored for 34 years he provided the first evidence of hereditary segregation and independent assortment In 1889 Hugo de Vries came up with the name "(pan)gene" for after postulating that particles are responsible for inheritance of characteristics Term "genetics" was coined by William Bateson in 1905. 19
  20. In 1928 Frederick Griffith proved the existence of a "transforming principle" involved in inheritance, which Avery, MacLeod and McCarty later (1944) identified as DNA. Edward Lawrie Tatum and George Wells Beadle developed the central dogma that genes code for proteins in 1941. The double helix structure of DNA was identified by James Watson and Francis Crick in 1953. 20
  21. In 1970 Hamilton Smiths lab discovered restriction enzymes that allowed DNA to be cut at specific places and separated out on an electrophoresis gel. • This enabled scientists to isolate genes from an organism's genome. DNA ligases, that join broken DNA together, had been discovered earlier in 1967 and by combining the two enzymes it was possible to "cut and paste" DNA sequences to create recombinant DNA. Plasmids, discovered in 1952, became important tools for transferring information between cells and replicating DNA sequences. 21
  22. Frederick Sanger developed a method for sequencing DNA in 1977, greatly increasing the genetic information available to researchers Polymerase chain reaction (PCR), developed by Kary Mullis in 1983, allowed small sections of DNA to be amplified and aided identification and isolation of genetic material Artificial competence was induced in Escherichia coli in 1970 when Morton Mandel and Akiko Higa showed that it could take up bacteriophage λ after treatment with calcium chloride solution (CaCl2). 22
  23. Two years later, Stanley Cohen showed that CaCl2 treatment was also effective for uptake of plasmid DNA. Transformation using electroporation was developed in the late 1980s, increasing the efficiency and bacterial range In 1972 Paul Berg utilised restriction enzymes and DNA ligases to create the first recombinant DNA molecules. 23
  24. • Herbert Boyer and Stanley N. Cohen took Bergs work a step further and introduced recombinant DNA into an bacterial cell. In 1981 the laboratories of Frank Ruddle, Frank Constantini and Elizabeth Lacy injected purified DNA into a single-cell mouse embryo and showed transmission of the genetic material to subsequent generations. On June 19, 2013 the leaders of three research teams who originated the technology, Robert T. Fraley of Monsanto; Marc VanMontagu of Ghent University in Belgium and founder of Plant Genetic Systems and CropDesign ; and Mary-Dell Chilton ofWashington University in St. Louis and Syngenta were awarded with the World Food Prize Gordon, J.; Ruddle, F. (1981). "Integration and stable germ line transmission 24 of genes injected into mouse pronuclei". Science 214 (4526): 1244.
  25. The first recorded knockout mouse was created by Mario R. Capecchi, Martin Evans and Oliver Smithies in 1989. They are used to study gene function and make useful models of human diseases. In 1992 onco-mice with tumor suppressor genes knocked out were generated. Creating Knockout rats are much harder and has only been possible since 2003 Bacteria synthesising human insulin were developed in 1979, being used as a treatment for the first time in 1982 Zan, Y; Haag, J. D.; Chen, K. S.; Shepel, L. A.; Wigington, D; Wang, Y. R.; Hu, R; Lopez-Guajardo, C. C.; Brose, H. L.; Porter, K. I.; Leonard, R. A.; Hitt, A. A.; Schommer, S. L.; Elegbede, A. F.; Gould, M. N. (2003). “Production of knockout rats using ENU mutagenesis and a yeast-based screening 25 assay". Nature Biotechnology 21(6): 645–51.
  26. 26 In 1988 the first human antibodies were produced in plants. The first animal to synthesise transgenic proteins in their milk were mice, engineered to produce human tissue plasminogen activator. With the discovery of microRNA in 1993 came the possibility of using RNA interference to silence an organisms endogenous genes - Peng, S. (2006). "A transgenic approach for RNA interference-based genetic screening in mice". Proceedings of the National Academy of Sciences 103 (7): 2252–2220. - Vaucheret, H.; Chupeau, Y. (2011). plant miRNAs regulate gene expression in animals Cell Research 22 (1): 3–5.
  27. 27  Improved our understanding of genetics.  His research helped to make organ transplantations possible. Dr. Bernard Amos 27
  28. • His work cloned frogs laid the foundations for somatic cell nuclear transfer, the application of which led to Dolly the sheep. 28 John Gurdon 28
  29. Worked out the Structure of Proteins. 29 Linus Pauling 29
  30. 30 “The Father of Cloning” Hans Spermann 30
  31. 31 “The Father of Genetics” Gregor Mendel 31
  32. • He noticed that there is a pattern in the 4 bases: Adenine, Guanine, Cytosine and Thymine. 32 • A=T and G=C. Erwin Chargaff 32
  33. In 1973 created a transgenic mouse by introducing foreign DNA into its embryo, making it the world’s first transgenic animal. 33 Rudolf Jaenisch 33
  35. GENERAL APPLICATIONS OF GENETIC ENGINEERING ][38] 35 Eg: transgenic plants produce natural pesticide to resist to pest 35
  36. Engineered Mammals A monkey named ANDi, for "inserted DNA", in a picture released in January 2001. ANDi was born in October 2000 at the Oregon Health Science University after receiving an extra bit of genetic material to become the world's first genetically modified non-human primate 36 36
  37. 37 Cloning Dolly • Sheep A: donate body cell nucleus • Sheep B: donate an egg cell without nucleus • Sheep C: surrogate mother A B C Dolly 37 Who’s its mother?
  39. GENETICS & ORAL HEALTH 39 39 Craniofacial & Tooth morphogenesis Agenesis GENETICS Dental caries Periodontistis Cleft lip & Palate Genetic disorders / Abnormalities Oral Cancer Malocclusion Behavorial Genetics Pharmacogenetics
  40. TECHNIQUES OF GENETIC ENGINEERING Tools and techniques Methods in recombinant DNA technology Genetically modified organisms Genetic treatments 40 40
  41. 41 TECHNIQUES OF GENETIC ENGINEERING Methods in recombinant DNA technology Genetically modified organisms Genetic treatments 41
  42.  DNA: The Raw Material – Heat-denatured DNA • DNA strands separate if heated to just below boiling • Exposes nucleotides • Can be slowly cooled and strands will renature 42 42
  43. Restriction Endo-nucleases • Enzymes that can clip strands of DNA crosswise at selected positions • Each has a known sequence of 4 to 10 pairs as its target • Can recognize and clip at palindromes 43 43
  44. • Can be used to cut DNA in to smaller pieces for further study or to remove and insert sequences. • Can make a blunt cut or a “sticky end” • The pieces of DNA produced are called restriction fragments. • Differences in the cutting pattern of specific restriction endonucleases give rise to restriction fragments of differing lengths-restriction fragment length polymorphisms. 44 44
  45. 45 45
  46.  Ligase and Reverse Transcriptase • Ligase: Enzyme necessary to seal sticky ends together • Reverse transcriptase: enzyme that is used when converting RNA into DNA. 46 46
  47. 47 47
  48. ANALYSIS OF DNA Gel electrophoresis Polymerase Chain Reaction 48 48
  49. 49 49  Gel electrophoresis: produces a readable pattern of DNA fragments
  50. GEL ELCTROPHORESIS • APPLICATIONS: Estimation of the size of DNA molecules following restriction enzyme digestion, e.g. in restriction mapping of cloned DNA. Analysis of PCR products, e.g. in molecular genetic diagnosis or genetic fingerprinting. 50 50
  51. 51 51 • Some techniques to analyze DNA and RNA are limited by the small amounts of test nucleic acid available • Polymerase chain reaction (PCR) rapidly increases the amount of DNA in a sample • So sensitive- could detect cancer from a single cell • Can replicate a target DNA from a few copies to billions in a few hours
  52. 52 52
  53. 53 53 Three Basic Steps that Cycle • Denaturation – Heat to 94°C to separate in to two strands – Cool to between 50°C and 65°C • Priming – Primers added in a concentration that favors binding to the complementary strand of test DNA – Prepares the two strands (amplicons) for synthesis • Extension – 72°C – DNA polymerase and nucleotides are added – Polymerases extend the molecule • The amplified DNA can then be analyzed
  54. 54 54 • Relative sizes of nucleic acids usually denoted by the number of base pairs (bp) they contain. • DNA Sequencing: Determining the Exact Genetic Code – Most detailed information comes from the actual order and types of bases- DNA sequencing – Most common technique: Sanger DNA sequence technique
  55. 55 55
  56. 56 56 • Two different nucleic acids can hybridize by uniting at their complementary regions. • Gene probes: specially formulated oligonucleotide tracers – Short stretch of DNA of a known sequence – Will base-pair with a stretch of DNA with a complementary sequence if one exists in the test sample • Can detect specific nucleotide sequences in unknown samples. • Probes carry reporter molecules (such as radioactive or luminescent labels) so they can be visualized. • Southern blot- a type of hybridization technique
  57. 57 57 • Southern blotting involves the transfer of DNA from a gel to a membrane, followed by detection of specific sequences by hybridization with a labeled probe. • Northern blotting, RNA is run on a gel. • Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection proteins of interest using antibodies.
  58. 58 FIGURE 21: Southern blot: Identifying Specific DNA Fragments (Edward Southern--the pioneer) or gentle vacuum pressure Drying or exposure to UV light Probes: Isotope or chemical Gel is soaked in alkali buffer to denature DNA
  59. Northern blotting is similar to Southern blotting, but involves the transfer of RNA from a gel to a membrane RNA 59
  60. Northern blotting: Measuring gene activity Poly(A)+ RNA: from rat tissues Probe: G3PDH (glyceraldehyde-3-phosphate dehydrogenase) 60
  61. Western blotting 61 • Western blotting entails separation of proteins on an SDS gel, transfer to a nitrocellulose membrane, and detection proteins of interest using antibodies. wikipedia
  62. Western blot 62
  63. Blotting Methods 63 • Antibodies can recognize the protein of interest or an epitope tag. • epitope tag – A short peptide sequence that encodes a recognition site (“epitope”) for an antibody, typically fused to a protein of interest for detection or purification by the antibody. Human influenza hemagglutinin (HA): YPYDVPDYA The HA tag is derived from the HA-molecule corresponding to amino acids 98-106 has been extensively used as a general epitope tag in expression vectors.
  64. 64 64
  65. 65 65 • Probes applied to intact cells • Observed microscopically for the presence and location of specific genetic marker sequences • Effective way to locate genes on chromosomes
  66. • Gene expression array are used to detect the level of all the expressed genes in an experimental sample. • SNP arrays permit genome-wide genotyping of single nucleotide polymorphisms. =>use allele-specific oligonucledtide probe • Array comparative genome hybridization (array-CGH) allows the detection of copy number changes in any DNA sequence compared between two samples. 66
  67. DNA 67 Microarrays • DNA microarrays comprise known DNA sequences spotted or synthesized on a small chip. Microarrays show the levels of all the expressed genes in an experimental sample.
  68. 68 TECHNIQUES OF GENETIC ENGINEERING Tools and techniques Genetically modified organisms Genetic treatments 68
  69. 69 69 Methods in Recombinant DNA Technology • Primary intent of recombinant DNA technology- deliberately remove genetic material from one organism and combine it with that of a different organism. • Form genetic clones – Gene is selected – Excise gene – Isolate gene – Insert gene into a vector – Vector inserts DNA into a cloning host
  70. 70 70 Methods in Recombinant DNA Technology
  71. 71 71 Technical Aspects of Recombinant DNA and Gene Cloning • Strategies for obtaining genes in an isolated state – DNA removed from cells, separated into fragments, inserted into a vector, and cloned; then undergo Southern blotting and probed – Gene can be synthesized from isolated mRNA transcripts – Gene can be amplified using PCR • Once isolated, genes can be maintained in a cloning host and vector (genomic library)
  72. 72 72 Characteristics of Cloning Vectors • Capable of carrying a significant piece of the donor DNA • Readily accepted by the cloning host • Must have a promoter in front of the cloned gene • Vectors (such as plasmids and bacteriophages) should have three important attributes: – An origin of replication somewhere on the vector – Must accept DNA of the desired size – Contain a gene that confers drug resistance to their cloning host
  73. 73 73
  74. 74 74 Characteristics of Cloning Hosts
  75. 75 APPLICATIONS OF GENETIC ENGINEERING Tools and techniques Methods in recombinant DNA technology Genetically modified organisms 75
  76. 76 76 TREATMENT OF GENETIC DISEASE • Conventional approach • Gene Therapy
  77. CONVENTIONAL APPROACH 77 77 OF TREATMENT • Enzyme induction by drugs • Replacement of deficient enzymes / proteins • Replacement of deficient vitamin / co-enzyme • Replacement of deficient product • Substrate restriction in diet • Drug therapy • Drug avoidance • Replacement of diseased tissue • Removal of disease tissue
  78. Genomic medicine 78 use of genotypic analysis (DNA testing) to enhance quality of medical care, including 78 - presymptomatic identification - preventive intervention - selection of pharmacotherapy - design of medical care
  79. 79 79 GENE THERAPY Replacement of a deficient gene / gene product or correction of an abnormal gene. 2 TYPES: i. Germ-line gene therapy – changes will be passed on to subsequent generations ii. Somatic Cell gene therapy – changes will not be passed on to future generations
  80. Gene Therapy • Gene transfer for the purpose of treating human disease. • Transfer of new genetic material as well as manipulation of existing genetic material. (Genetic engineering) in vivo ex vivo 80
  81. 81
  82. Gene therapy Potential Uses • Treatment of recurrent disease • Adjuvant treatment • Localized distant metastatic disease 82
  83. Delivery systems / vectors  Non – viral - Electropolation - DEAE-dextran - Calcium phosphate - Liposomes -Naked DNA  Viruses Retroviruses Adenoviruses Adeno-associated viruses Herpes virus Gene therapy 83
  84. Gene therapy 84
  85. Gene therapy in dentistry 1. Bone repair • Mesenchymal stem –cell mediated gene therapy (BMPs) • Using adenoviral vector • Transfer of Platelet derivative growth factor (PDGF) • Bone sialoprotein delivery (in-vivo) 2. Salivary glands • Irreversible salivary gland dysfunction Gene therapy 85
  86. Gene therapy • Adenovirus encoding human AQP1 (water channel protein) – irradiated salivary gland hyposalivation. • Autoimmune diseases Sjogren syndrome : cytokines inflammation adeno-associated virus, AAV, serotype2 IL-10 transfer using recombinant AAV2 vector – salivary glands hyposalivation . 86
  87. Gene therapy 87 87
  88. • Gene therapeutics Gene therapy 88 local (exocrine) systemic (endocrine) (oral, pharyngeal, (single protein & esophageal) deficiency) Eg mucosal cadidiasis Eg hGH • Azole resistant • Recombinant adenoviral vector encoding H3
  89. Gene therapy • Pain Virus vector – mediated transfer of genes encoding opiate peptides peripheral & central neurons Anti-noceptive effects 89 Direct gene delivery – articular surface TMJ
  90. • Keratinocytes Gene therapy – systemic human aplipoprotein E, factor IX, growth hormone and IL-10 into bloodstream. • DNA vaccinations Gene therapy 90
  91. Gene therapy • Gene gun-based DNA vaccination against infectious diseases – oral mucosa (Wang J 2003) • Caries vaccine 91
  92. • Subunit Vaccines Gene therapy - synthetic peptide vaccines - recombinant vaccines • Conjugate Vaccines • Routes to Protective Responses - oral - intranasal - tonsillar - rectal 92 • Adjuvants and Delivery Systems Cholera & E coli, microcapsules, liposomes
  93. Human applications Gene therapy 93 - Active immunization ( 7 trials) - Passive immunization ( cow’s milk, chicken eggs, transgenic plant antibody)
  94. Gene therapy 94 Future Strategies of Gene Therapy in Preventing Periodontal Diseases • Gene Therapeutics-Periodontal Vaccination • Genetic Approach to Biofilm Antibiotic Resistance • An In vivo Gene Transfer by Electroporation for Alveolar Remodelling • Tight Adherence Gene for the Control of Periodontal Disease Progression • Antimicrobial Gene Therapy to Control Disease Progression 94
  95. Gene therapy 95 95
  96. • AIDS vaccine • HPV vaccine • HSV vaccine  Head & neck cancer Gene therapy 96
  97. Current gene therapies for cancer 97 Mechanism Goal Development stage Oncogene down-regulation therapy Delete defective gene Inhibit tumor cell growth Pre-clinical Gene addition therapy Add tumor suppressor gene Kill tumor cell Clinical trial Anti-sense RNA Abrogate genes stimulating tumor growth Inhibit tumor cell growth Clinical trial Immunothera py Enhance immune surveillance Enhance immunogenic ity of tumor cell Clinical trial
  98. Anti-angiogenesis therapy Transfer gene to tumor cells to block angiogenesis Inhibit tumor progression Pre-clinical Drug resistance gene therapy Transfer cytoprotectiv e gene Decrease toxicity of chemotherap y Clinical trial Tumor-cell killing viruses Introduce viruses that destroy tumor cells as part of replication cycle Kill tumor cells Pre-clinical Suicide gene therapy Transfer gene encoding pro-drug activating enzyme Kill tumor cell & enhance chemotherap y Clinical trial 98
  99. 99 HUMAN GENOME 99 PROJECT Objectives: i. Sequencing of human genomes ii. Mapping of human inherited diseases iii. Development of new DNA technologies iv. Development of bio-informatics v. Comparitive Genomics vi. Functional Genomics
  100. 100 TISSUE ENGINEERING • Tissue Engineering is a general name of biomedical fields to enable cells to enhance their proliferation, differentiation, and morphological organization for induction of tissue regeneration, resulting in regenerative medical therapy of diseases. 100
  101. 101 Stem cells in regenerative medicine • A stem cell is defined as a cell that can continuously produce unaltered daughters and, furthermore, has the ability to generate cells with different and more restricted properties. • These cells can either multiply (progenitors or transit amplifying cells) or be committed to terminal differentiation. • Stem cells are self-renewing and thus can generate any tissue for a lifetime. • This is a key property for a successful therapy. 101
  102. 102 102
  103. 103 103
  104. 104 104
  105. 105 105
  106. 106 106
  107. 107 GENETIC COUNSELLING A process of communication and education which addresses concerns relating to the development and / or transmission of a hereditary disorder. STEPS IN GENETIC COUNSELLING 107 - Diagnosis - Risk assessment - Communication - Discussion of options - Long term contact & support
  108. 108 108 DIAGNOSIS • History • Examination • Investigation • Only when accurate diagnosis is possible • When etiological heterogeneity is present
  109. 109 RISK ASSESSMENT The good side of the coin should also be emphasized 109 ARBITRARY GUIDE  1 in 10 - High risk  1 in 20 - Low risk  Intermediate values - Moderate risk
  110. 110 LONG TERM CONTACT & 110 SUPPORT • Counselling centers should maintain informal contact with families through a network of genetic associates • Genetics registers provide a useful means in ensuring effective contact
  111. 111 NEONATAL SCREENING 111 To prevent subsequent morbidity POPULATION CARRIER SCREENING The branch of medical genetics which is concerned with screening and the prevention of genetic disease on a population basis is known as community genetics.
  112. PRENATAL DIAGNOSIS 112 Ability to detect abnormality in an unborn child. 112 TECHNIQUES I. Non invasive - Maternal Serum screening - Ultra sound II. Invasive - Amniocentesis - Chorionic Villus Sampling
  113. INDICATIONS FOR 113 113 PRENATAL DIAGNOSIS • Advanced maternal age • Previous child with a genetic abnormality • Family History of - Chromosome abnormality - Single gene disorder - Neural tube defect - Other congenital structural abnormalities • Abnormalities identified in pregnancy Eg. Poor fetal growth
  114. 114 114 • Other High risk factors - Parental Consanguinity - Poor obstetric history Eg: Recurrent miscarriages Previous unexplained still birth - Maternal illness Eg: Poorly controlled IDDM Maternal epilepsy Treatment with Sodium Valproate
  115. IDENTIFY GENETIC DISEASE 1. Build the pedigree 2. Analyse 3. Risk of recurrence 4. Decision 115
  116. Role of dentist as genetic counselor • Oral manifestations • Correct identification • Diagnosis • Referral • Suggestion • Screening for dental diseases DNA probes 116
  117. Future prospects 117 • Bioengineering • Nanodentistry • Biomimetics • Molecular Epidemiology ( Variation Genetics )
  118. 118 118
  119. 119 Genetic engineering Enabling technology Cutting,modifying and joining DNA molecules enzymes Generation of DNA fragments Restriction enzyme DNA Ligase Joining to a vector or DNA Molecule Introduction into the host cell Selection of desired sequence Arose from Gene cloning Recombinant DNA Molecular cloning Pure science, Biotechnology, Medicine, Dentistry Legal and ethical considerations Microbial & Molecular genetics In 1972 Stanford University Is also known as Has application in But raises some was first achieved Is an That involves using Such as Requires four steps Can be used for used for
  120. CONCLUSION 120 • Biotechnology as a fast developing technology as well as science , has already shown its impact on different aspects of day-to-day human life such as public health pharmaceuticals, food and agriculture industries, bioenergetics and information technology. 120
  121. 121 • As it has potential to ensure food security, dramatically reduce hunger and malnutrition and reduce rural poverty , particularly in developing countries , Now it is very clear that biotechnology is the key technology for the 21st century and the science of the future. 121
  122. References 1. Colin EC. What Is Genetics? In: Colin EC. Elements of Genetics. 3rd ed. New York: McGraw Hill Book Company, Inc; 1956. p. 1-3. 2. Auden WH. History of Human Genetics. In: Motulsky V. Human Genetics, Problems and Approaches. 3rd ed. New York: Springer; 1997. p. 1-22. 3. Dhar PK. Genetics in pediatric dentistry. In: Tandon S. Textbook of Pedodontics. Ist ed. Hyderabad: Paras Medical Publisher; 2001. p. 614-622. 4. Baeudet AL. Genetics and Disease. In: Fauci, Braunwald, Isselbacher, Wilson, Martin, Hauser, Longo. Harrison’s Principles of Internal Medicine. Vol 2. 14th ed. New York: McGraw Hill Companies, Inc; 1998. p. 365-395.
  123. 5 Tortora GJ, Grabowski SR. Cellular level of organization. 10th ed. John wiley & sons, USA. 84-97. 6 Pashayan HM, Feingold M. Selected syndromes in pedodontics. White GE. Clinical oral pediatrics. Quintessence International. Tokyo. 1981. ed. 73-80. 7 Joerde LB, Carey JC, White RL. Genetic Variation: Its Origin and Detection. In: Joerde LB, Carey JC, White RL. Medical Genetics. Ist ed. Toronto: Mosby; 1995. p. 30-56. 8 Tencate AR et al. Development of tooth and its supporting structures. In: Nanci A. Tencate’s oral histology. 8thed. Mosby. 2003: 79-110. 9 Tandon S, Bhat S. Developing dentition and its disturbances. In: Tandon s. Textbook of pedodontics. Ed. Paras publishing . 2001. 85- 105.
  124. 10. Shafer. Textbook of oral pathology. 4th ed. Harcourt Asia PTE Ltd.. 1999. 11 Anderson M. Risk Assessment and Epidemiology of Dental Caries: Review of Literature. Pediatr Dent 2002; 24(5): 377- 385. 12 Sofaer JA. Host genes and dental caries. Br Dent J 1993; 175: 403-409. 13 Shuler Cf. Inherited risks for susceptibility to dental caries. J Dent Educ 2001; 65(10): 1038-1045. 14. Hart TC & Kornman KC. Genetic factors in pathogenesis of periodontitis. Periodont 2000 1997; 14: 202-15. 15 Michalowicz. Genetic & heritable risk factors in periodontal disease. J Periodontol 1994; 65: 479-88.
  125. 16 Cobourne MT. the complex genetics of cleft lip & palate. Euro J Ortho 2004; 26: 7-16. 17 Mossey PA et al. The heritability of malocclusion. The influence of genetics in malocclusion. Br J Ortho 1999; 26: 195-203. 18 Partridge M. Oral cancer: the genetic basis of disease. Dent Update 2000; 27: 242-248. 19 Carrozzo M. Hepatitis C virus-associated oral lichen planus: is the geographical heterogeneity related to HLA-DR6?J Oral Pathol Med. 2005 Apr;34(4):204-8. 20 Lawson CAet al. Analysis of the insertion/deletion related polymorphism within T cell antigen receptor beta variable genes in primary Sjogren's syndrome.Ann Rheum Dis. 2005 Mar;64(3):468-70.
  126. 24 Campisi G. HPV DNA in clinically different variants of oral leukoplakia and lichen planus. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2004 Dec;98(6):705-11. 25 Xi S etal. Gene therapy for the treatment of oral squamous cell carcinoma. J Dent Res 2003; 82: 11-16. 26 Baum JB. The impact of gene therapy on dentistry. JADA 2002; 133: 35-44. 27 Wang J et al. Predominant cell-mediated immunity in the oral mucosa: gene gun-based vaccination against infectious diseases. J Dermatol Sci. 2003 May;31(3):203-10. 28 Slavkin HC. And the next 50 years. The future of DNA technology in oral medicine. JPHD 1996; 56: 278-85.
  127. 29. 30. 31. _oralcavity2_p.htm 32 . 33. 34 35 .Faiez N. Genetics. DCNA. 1975. 1-150. 36. Wise GE. Cell and molecular biology of tooth eruption. In: Davidowitch Z. Biologic mechanisms of tooth eruption. Ed. Harvard society. Boston. 1998. 1-7.
  128. 37. Griffiths, Anthony J. F.; Miller, Jeffrey H.; Suzuki, David T.; Lewontin, Richard C.; Gelbart, William M., eds. (2000). "Genetics and the Organism: Introduction". An Introduction to Genetic Analysis (7th ed.). New York: W. H. Freeman. 38. Book : Biotechnology & Genetic engineering (Kathy wilson peacock) 2010,Edi:1 : Page No. 4 (Chapter 1) 39. D. L. Hartl and V. Orel (1992). "What Did Gregor Mendel Think He Discovered?".Genetics 131 (2): 245–25 40. Zambryski, P.; Joos, H.; Genetello, C.; Leemans, J.; Montagu, M. V.; Schell, J. (1983). "Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity". The EMBO Journal 2 (12): 2143–2150. 128

Notas do Editor

  1. In 1907 a bacterium that caused plant tumors, Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor inducing agent was found to be a DNA plasmid called the Ti plasmid By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants
  2. By removing the genes in the plasmid that caused the tumor and adding in novel genes researchers were able to infect plants with A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants
  3. Genetic engineering has been used to produce proteins derived from humans and other sources in organisms that normally cannot synthesise these proteins. Bacteria synthesising human insulin were developed in 1979, being used as a treatment for the first time in 1982.[48] In 1988 the first human antibodies were produced in plants.[In 1997avidin, an egg protein, was expressed in a plant with the intention of extracting, purifying and selling it.[49] The first transgenic livestock were produced in 1985,[50] by micro injecting foreign DNA into rabbits, sheep and pigs eggs.[51] The first animal to synthesise transgenic proteins in their milk were mice,[52] engineered to produce human tissue plasminogen activator.[53] This technology has now been applied to other sheep, pigs, cows and other livestock.[52] With the discovery of microRNA in 1993 came the possibility of using RNA interference to silence an organisms endogenous genes. Craig C. Mello and Andrew Fire discovered a silencing effect in 1998 through injection of double stranded RNA into C. Elegans . Using genetic engineering the microRNA can be expressed long term, permanently silencing the target genes. In 2002 stable gene silencing was induced in mammalian cells,[and in 2005 this was accomplished in a whole mouse.[In 2007 papers were released where insect and nematode genes that formed microRNA were put into plants, resulting in gene silencing of the pest when they ingested the transgenic plant.[58]
  4. The development of genetic engineering technology led to concerns in the scientific community about potential risks. The development of a regulatory framework concerning genetic engineering began in 1975, at Asilomar, California. The Asilomar meeting recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology.[30] The Asilomar recommendations were voluntary, but in 1976 the US National Institute of Health (NIH) formed a recombinant DNA advisory committee.[31] This was followed by other regulatory offices (the United States Department of Agriculture (USDA), Environmental Protection Agency (EPA) and Food and Drug Administration (FDA)), effectively making all recombinant DNA research tightly regulated in the USA.[In 1982 the Organization for Economic Co-operation and Development (OECD) released a report into the potential hazards of releasing genetically modified organisms into the environment as the first transgenic plants were being developed.[33] As the technology improved and genetically organisms moved from model organisms to potential commercial products the USA established a committee at theOffice of Science and Technology (OSTP) to develop mechanisms to regulate the developing technology.[32] In 1986 the OSTP assigned regulatory approval of genetically modified plants in the US to the USDA, FDA and EPA.[34] In the late 1980s and early 1990s, guidance on assessing the safety of genetically engineered plants and food emerged from organizations including the FAO and WHO.[35][36][37 WHO (1987): Principles for the Safety Assessment of Food Additives and Contaminants in Food, Environmental Health Criteria 70. World Health Organization, Geneva Jump up^ WHO (1991): Strategies for assessing the safety of foods produced by biotechnology, Report of a Joint FAO/WHO Consultation. World Health Organization, Geneva Jump up^ WHO (1993): Health aspects of marker genes in genetically modified plants, Report of a WHO Workshop. World Health Organization, Geneva Jump up^ WHO (1995): Application of the principle of substantial equivalence to the safety evaluation of foods or food components from plants derived by modern biotechnology, Report of a WHO Workshop. World Health Organization, Geneva n 1976 Genentech, the first genetic engineering company was founded by Herbert Boyer and Robert Swanson and a year later and the company produced a human protein (somatostatin) in E.coli. Genentech announced the production of genetically engineered human insulin in 1978.[59] In 1980, the U.S. Supreme Court in the Diamond v. Chakrabarty case ruled that genetically altered life could be patented.[60] The insulin produced by bacteria, branded humulin, was approved for release by the Food and Drug Administration in 1982.[61] In 1983, a biotech company, Advanced Genetic Sciences (AGS) applied for U.S. government authorization to perform field tests with the ice-minus strain of P. syringae to protect crops from frost, but environmental groups and protestors delayed the field tests for four years with legal challenges.[62] In 1987, the ice-minus strain of P. syringae became the first genetically modified organism (GMO) to be released into the environment[63] when a strawberry field and a potato field in California were sprayed with it.[64] Both test fields were attacked by activist groups the night before the tests occurred: "The world's first trial site attracted the world's first field trasher".[63] The first field trials of genetically engineered plants occurred in France and the USA in 1986, tobacco plants were engineered to be resistant to herbicides.[65] The People’s Republic of China was the first country to commercialize transgenic plants, introducing a virus-resistant tobacco in 1992.[66] In 1994 Calgene attained approval to commercially release the Flavr Savr tomato, a tomato engineered to have a longer shelf life.[67] In 1994, the European Union approved tobacco engineered to be resistant to the herbicidebromoxynil, making it the first genetically engineered crop commercialized in Europe.[68] In 1995, Bt Potato was approved safe by the Environmental Protection Agency, after having been approved by the FDA, making it the first pesticide producing crop to be approved in the USA.[69] By 2010, according to the annual ISAAA brief: "While 29 countries planted commercialized biotech crops in 2010, an additional 31 countries, totaling 60 have granted regulatory approvals for biotech crops for import for food and feed use and for release into the environment since 1996.... A total of 1,045 approvals have been granted for 196 events (NB: an "event" is a specific genetic modification in a specific species) for 25 crops. Thus, biotech crops are accepted for import for food and feed use and for release into the environment in 60 countries, including major food importing countries like Japan, which do not plant biotech crops. Of the 60 countries that have granted approvals for biotech crops, USA tops the list followed by Japan, Canada, Mexico, South Korea, Australia, the Philippines, New Zealand, the European Union, and Taiwan. Maize has the most events approved (65) followed by cotton (39), canola (15), potato and soybean (14 each). The event that has received regulatory approval in most countries is herbicide tolerant soybean event GTS-40-3-2 with 25 approvals (EU=27 counted as 1 approval only), followed by insect resistant maize MON810 with 23 approvals, herbicide tolerant maize NK603 with 22 approvals each, and insect resistant cotton (MON1445) with 14 approvals worldwide."
  5. strand that contains the same series of nitrogenous bases regardless from which direction the strand is analyzed.