<h5 id="41-dna-fingerprinting">4.1 DNA Fingerprinting</h5> <ul> <li>The chemical structure of DNA is identical in all individuals, made up of four bases: A, G, C, and T. The arrangement of these bases along the DNA strands is unique to each individual.</li> <li>More than 90% of the human genome is non-coding and contains short tandem repeats (VNTR) that show high polymorphism. These VNTRs are inherited and form the basis of DNA fingerprinting.</li> <li>DNA fingerprinting can be performed using restriction digestion of DNA, known as Restriction Fragment Length Polymorphism (RFLP), which generates unique patterns for genetic analysis and identification.</li> <li>Southern hybridization using VNTR probes produces a pattern of bands that are specific to every individual, used in forensics and paternity disputes.</li> <li>Applications of DNA fingerprinting include paternity and maternity cases, criminal identification and forensic studies, evolutionary biology studies, identifying DNA patterns in inherited disorders, and personal identification using DNA profiles.</li> </ul> <p>======</p> <h5 id="42-transgenic-organism">4.2 Transgenic Organism</h5> <ul> <li>Transgenic organisms are produced through the process of transgenesis.</li> <li>Transgenesis involves the introduction of new gene segments into the genome of an organism.</li> <li>The organisms that carry the transgene are called transgenic organisms or genetically modified organisms (GMOs).</li> <li>These organisms are not naturally found in the environment.</li> <li>An example of a transgenic plant is Bt Cotton, and an example of a transgenic animal is Rosie, the cow.</li> <li>Transgenic organisms are created for the benefit of human beings.</li> </ul> <p>======</p> <h5 id="421-historical-background">4.2.1 Historical background</h5> <ul> <li>The first genetically modified organism was a bacterium created by Herbert Boyer and Stanley Cohen in 1973.</li> <li>The first transgenic animal (transgenic mice) were engineered by Rudolf Jaenisch and Beatrice Mintz in 1974.</li> <li>The first genetically modified food crop approved by the USFDA was the Flavr Savr tomato, released in 1994.</li> <li>The concept of transgenic organisms has been advanced through key events outlined in the timeline in Box 1.</li> <li>Reference: Rangel, G. (2015). From Corgis to Corn: a Brief Look at the Long History of GMO Technology. Science in the News.</li> </ul> <p>======</p> <h5 id="422-production-of-transgenic-organisms">4.2.2 Production of transgenic organisms</h5> <ul> <li>Transgenic plants are genetically modified plants with a genome altered through genetic engineering.</li> <li>The two main techniques for gene transfer are vector-mediated (indirect) and vector-less (direct).</li> <li>Vector-mediated gene transfer includes bacteria mediated transfer using Agrobacterium tumefaciens, which has the natural ability to transfer its own DNA to plant genomes.</li> <li>Vector-less gene transfer includes particle bombardment gene transfer, a method that delivers foreign DNA into plant cells using a particle gun.</li> <li>Transgenic animals are produced using various genetic engineering techniques, including DNA pronuclear microinjection, embryonic stem cell-mediated gene transfer, and retrovirus-mediated gene transfer.</li> </ul> <p>======</p> <h5 id="423-applications-of-transgenic-organisms">4.2.3 Applications of Transgenic organisms</h5> <ul> <li>Transgenic animals are genetically manipulated organisms used for research purposes.</li> <li>They are used to study normal human physiology, development, and diseases.</li> <li>Transgenic animals can be manipulated to develop symptoms of diseases, such as cancer and Alzheimer’s.</li> <li>This allows researchers to understand the function of genes involved in various diseases.</li> <li>Transgenic animals are also used for toxicity testing of vaccines, drugs, and chemicals before they can be used on humans.</li> </ul> <p>======</p> <h5 id="424-concern-over-gmos">4.2.4 Concern over GMOs</h5> <ul> <li>The primary concern over GMOs is the potential risk to human health and the environment.</li> <li>Transgenic crops may produce proteins that can cause allergic reactions in humans.</li> <li>The use of viral vectors and promoters in GMOs can lead to viral infections in humans.</li> <li>The improved characteristics of GMOs can make them invasive, impacting native or wild type species.</li> <li>Variable insertion of transgenes can lead to unpredictable or non-target effects.</li> <li>India has established biosafety regulations for the production and use of GMOs, with the Genetic Engineering Approval Committee (GEAC) regulating their manufacture, use, import, export, and storage.</li> </ul> <p>======</p> <h5 id="43-gene-therapy">4.3 Gene Therapy</h5> <ul> <li>Gene therapy is a technique designed to repair faulty genes in humans by introducing correct genetic material inside the cells.</li> <li>The obvious candidate diseases for gene therapy include severe combined immunodeficiency (SCID) and inherited genetic disorders such as cystic fibrosis, hemophilia, muscular dystrophy, etc.</li> <li>The first gene therapy was performed in 1990 in a four-year-old girl suffering from SCID caused due to the defect in the adenosine deaminase (ADA) coding gene.</li> <li>A functional copy of the ADA gene was inserted into a viral vector, which was then introduced into lymphocytes collected from the patient.</li> <li>These lymphocytes were reintroduced into the patient’s bloodstream, leading to an improvement in the patient’s immune system functioning, although the cure was not permanent.</li> </ul> <p>======</p> <h5 id="431-approaches-for-gene-therapy">4.3.1 Approaches for Gene Therapy</h5> <ul> <li>Gene therapy has three main approaches: Gene replacement or Gene addition, Gene inhibition, and Gene repair or Gene editing therapies.</li> <li>In Gene replacement or Gene addition therapy, a functional copy of the gene is delivered into the genome to replace the non-functional defective gene. This is used to cure diseases such as cystic fibrosis and cancer.</li> <li>In Gene inhibition, the function of a gene which causes a disease in the cell is inactivated. This approach is applicable in infectious diseases, cancer, and mutated genes whose products cause diseases.</li> <li>Gene repair or Gene editing is achieved by the CRISPR/Cas9 system, a recent technology that was adopted from the natural defense system of bacteria or archaea against viruses. This system offers an easy way to snip out mutated DNA and replace it with the correct sequence.</li> <li>The CRISPR/Cas9 system has the potential to treat several genetic disorders in the future.</li> </ul> <p>======</p> <h5 id="432-types-of-gene-therapies">4.3.2 Types of Gene Therapies</h5> <ul> <li> <p>Gene therapy can be classified into ex vivo and in vivo based on the gene delivery strategy. Ex vivo involves removing cells, introducing normal genes in culture, and reintroducing them into the patient. In vivo therapy introduces normal functional genes directly into the target cells or tissues.</p> </li> <li> <p>Based on the nature of target cells or tissues, gene therapy can be classified into somatic and germ-line. Somatic gene therapy introduces functional copies into the patient’s somatic cells, while germ-line gene therapy introduces genes into the germ cells, leading to heritable changes.</p> </li> <li> <p>There are three main functional strategies in gene therapy:</p> <ul> <li>Gene augmentation therapy (GAT): Adding a functional gene to the genome to replace a missing gene product.</li> <li>Gene inhibition therapy (GIT): Blocking the expression of dominant acting mutated genes using antisense RNA or other inhibition techniques.</li> <li>Gene targeting: Replacing a non-functional gene with a normal gene using homologous recombination.</li> </ul> </li> <li> <p>The possibility of correcting defective genes through these strategies is due to advancements in rDNA technology, gene transfer, and gene editing mechanisms.</p> </li> <li> <p>The target gene (desired DNA sequence) can be delivered using either viral (direct gene transfer) or non-viral (indirect gene transfer) methods, as studied in previous chapters.</p> </li> </ul> <p>======</p> <h5 id="433-merits-and-demerits-of-gene-therapy">4.3.3 Merits and Demerits of Gene Therapy</h5> <ul> <li>Gene therapy can potentially cure inherited diseases with little hope from conventional treatments.</li> <li>Uncontrolled expression of the therapeutic gene is a concern for efficient gene therapy.</li> <li>Introduction of the therapeutic gene in non-target cells or its random integration in the host genome can lead to tumors.</li> <li>Frequent administration of the therapeutic gene may be required as gene therapy is short-lived.</li> <li>Host rejection of the therapeutic gene or the viral vector can stimulate the host’s immune system, and gene therapy can be costly.</li> <li>Gene therapy is currently effective for single-gene defects but not for multigene disorders.</li> </ul> <p>======</p> <h5 id="434-ethical-issues">4.3.4 Ethical Issues</h5> <ul> <li>Bioethics involves assessing risks and moral implications of new techniques, with genetic modification of somatic cells approved but germ line gene therapy often debated.</li> <li>Ethical concerns around germ line gene therapy include: deciding its ethics, the potential for gene enhancement, unequal access to technology, and exploitation by developed/rich countries.</li> <li>Germ line gene therapy requires prenatal genetic testing, which can lead to increased abortions/foeticides, and may be used for gene enhancement with unpredictable outcomes.</li> <li>Strict regulations are needed to ensure gene therapy research is not misused for gene enhancement purposes.</li> <li>Gene therapy has been primarily used in patients for whom all other treatments have failed, and is often criticized for promising too much but delivering little.</li> <li>With constant efforts, gene therapy may become a common practice for treating genetic diseases with single gene defects.</li> </ul> <p>======</p> <h5 id="44-recombinant-vaccines">4.4 Recombinant Vaccines</h5> <ul> <li>A vaccine is a preparation of killed or weakened pathogen or their components given to elicit an immune response.</li> <li>Initially, vaccines were developed mostly by attenuation or inactivation of pathogens, but recombinant vaccines were developed to overcome potential concerns.</li> <li>There are three main types of recombinant vaccines: <ol> <li>Live genetically modified vaccines: These are developed by modifying the pathogen into a non-pathogenic organism through deletion or inactivation of one or more genes.</li> <li>Recombinant subunit vaccines: These contain only a part of the whole pathogenic organism, such as synthetic peptide or an expressed whole protein extracted from the pathogenic organism.</li> <li>DNA vaccines: For DNA vaccines, genes of interest (antigens) are identified and cloned in plasmids, which are then directly injected into the muscles of the animal model system.</li> </ol> </li> <li>RNA vaccines contain mRNA that is directly taken up by antigen presenting cells and other target cells, where the mRNA is expressed as properly folded and glycosylated protein that acts as an antigen.</li> <li>Recombinant and RNA vaccines have several advantages over other categories of vaccines, including high yield production, virus-free production process, and ability to respond rapidly to emerging viral variants.</li> </ul> <p>======</p> <h5 id="45-therapeutic-agentsmolecules-monoclonal-antibodies-insulin-and">4.5 Therapeutic Agents/Molecules: Monoclonal Antibodies, Insulin And</h5> <ul> <li>Recombinant DNA technology allows large-scale production of various human proteins for therapeutic use, including monoclonal antibodies, insulin, and growth hormone.</li> <li>Monoclonal antibodies are specific protein molecules produced by B cells, which can be exploited for in vitro purposes like therapeutic and diagnostic applications. They can be produced using hybridoma technology, where a short-lived antibody-secreting normal B cell is fused with immortal myeloma cells.</li> <li>Insulin is a hormone that maintains glucose homeostasis in the body. It consists of two chains: an A chain of 21 amino acids and a B chain of 30 amino acids, interconnected with disulphide bonds. Human insulin, porcine insulin, and bovine insulin are very similar, but minordifferences in the chemical structure can lead to an allergic response in some diabetic patients.</li> <li>Genetically engineered insulin was first marketed as Humulin in 1982, produced by inserting the insulin gene from a gene library into a bacterial plasmid of E. coli. The gene expression produced a fusion protein, which was later cleaved to produce the human insulin.</li> <li>Human growth hormone (HGH) containing 191 amino acids is a peptide hormone that promotes body growth. It can be produced using rDNA technology, similar to human insulin production. The production process for HGH involves synthesising the gene for HGH using HGH encoding mRNA as a template and a complementary DNA (cDNA) molecule.</li> </ul> <p>======</p>