Biotechnology: Application of Molecular Genetics Principles and Tools (Ch.21)

Biotechnology Overview

  • Definition of Biotechnology: Biotechnology is broadly defined as technologies that involve the use of living organisms or their products to benefit humans.
      - Historical Context: The origins of biotechnology date back several thousand years when humans began domesticating animals and plants for food production.

Molecular Genetics in Biotechnology

  • Advancements since the 1970s: Molecular genetics has provided innovative methods to utilize organisms for human benefits.
      - Genetically Modified Organisms (GMOs): These are organisms that have received genetic material through recombinant DNA technology.
      - Transgenic Organisms: An organism that integrates recombinant DNA from a different species into its genome is referred to as transgenic.

Applications of Microorganisms in Biotechnology

  • General Uses: Microorganisms play a vital role in biotechnology by benefiting humans.
      - Importance of Molecular Genetic Tools: These tools greatly enhance and influence the utilization of microorganisms in various applications.
      - Research Interest and Potential: The use of recombinant microorganisms is a significant area of research; however, it faces challenges like safety concerns and negative public perception.

Common Applications of Microorganisms (Table 21.1)

  • Production of Medicines: Examples include antibiotics and vitamins.

  • Synthesis of Human Insulin: Produced in recombinant E. coli.

  • Food Fermentation: Used in making cheese, yogurt, vinegar, wine, and beer.

  • Bioremediation: Involves the cleanup of environmental pollutants, such as petroleum hydrocarbons and synthetics challenging to degrade.

Medicines Derived from Recombinant Microorganisms (Table 21.2)

  • Insulin: A hormone that promotes glucose uptake used in diabetes treatment.

Chemistry of Insulin

  • Composition: Insulin consists of two polypeptide chains, referred to as the A and B chains.

  • Function: It regulates several physiological processes, particularly the uptake of glucose into fat and muscle cells, produced by β cells of the pancreas.
      - Diabetes Relation: Insulin-dependent diabetes results from defective β cells; patients cannot synthesize sufficient insulin.

Sources of Insulin

  • Traditional Sources: Historically sourced from cows and human cadavers.

  • Modern Source: Currently, recombinant bacteria are primarily used to produce insulin.

Natural Synthesis of Human Insulin Process

  • Preproinsulin: Initial stage of insulin synthesis.

  • Multistep Process: Involves several stages, including the cleavage of signal sequences, disulfide bond formation, and the removal of connecting polypeptides, leading to proinsulin and, subsequently, insulin.

Synthesis of Genetically Engineered Insulin in E. coli

  • Gene Insertion: Involves insertion of ampR and other components necessary for cloning the insulin gene into E. coli.

  • Cell Culture: Cells are cultured, leading to the production of both A and B chains of insulin.

  • Purification: Fusion proteins are isolated, and the A chain and B chain of insulin are purified.

  • Refolding: Processes are conducted to create active insulin through disulfide bond formation.

Genetically Modified Animals

  • Transgenic Animals: Production of these animals represents an exciting area in biotechnology, which offers considerable promise.
      - Public Acceptance: Success in this field is largely contingent upon public acceptance.

Gene Modification Techniques

  • Altering Genomes: Gene modification can result from gene editing or gene addition.
      - Gene Editing: Alters the sequence of a gene.
      - Gene Addition: Involves the insertion of a cloned gene into a cell's genome (e.g., gene knock-in in mice).

Gene Editing Techniques

  • Mutations and Their Importance: Analysis of mutations contributes valuable insights into normal genetic processes and can occur spontaneously or through mutagens.
      - Experimental Mutation Creation: Known as gene editing, it allows for precise alterations within cloned DNA or living cells.

CRISPR-Cas Technology

  • Mechanism: This technology enables gene editing using a system originally used by bacteria to combat bacteriophages.
      - Single Guide RNA (sgRNA): Researchers design sgRNA by linking tracrRNA and crRNA, allowing targeted gene editing by guiding the Cas9 enzyme.
      - Double-Strand Break: Cas9 creates a double-strand break at the target gene facilitated by sgRNA.

DNA Repair Post-Crispr Cut

  1. Nonhomologous End Joining (NHEJ): May lead to small deletions that inactivate genes.

  2. Homologous Recombination Repair (HRR): Requires donor DNA to introduce desired mutations.

Gene Addition Example: GloFish

  • Mechanism: Genes from other species (e.g., jellyfish) are added to zebrafish, creating a transgenic organism with fluorescent properties.

  • Historical Highlight: In 2003, GloFish became the first genetically modified organism marketed as a pet.

Gene Knockouts in Mice

  • Purpose: Understanding gene function and human disease through the creation of gene knockout mice using methods like CRISPR-Cas technology.

  • Inheritance Pattern: Heterozygous parental mice can produce homozygous offspring carrying gene knockouts.

  • Observable Phenotypes: Phenotypes of knockouts help reveal gene functions within specific tissues or developmental stages.

Use of Animal Models in Human Disease Research

  • Model Development: Engineered to exhibit gene mutations analogous to those found in human diseases; e.g., sickle cell disease involves multiple gene alterations and knockouts.

Transgenic Animals for Xenotransplantation

  • Engineering for Transplantation: Genetic modifications reduce immunogenicity and enhance the viability of organs for transplantation into humans.

Process of Expressing Human Genes in Animal Milk

  1. Insert the human gene into a plasmid vector near a milk-specific promoter.

  2. Inject this DNA into a sheep oocyte, integrating the gene into its genome.

  3. Implant the fertilized oocyte into the uterus of a female sheep.

  4. Resulting transgenic offspring produce the human protein in milk, which is subsequently purified.

Cloning and Stem Cell Technology

  • Reproductive Cloning: Produces genetically identical individuals and is applied to plants and mammals.
      - Historical Milestone: Dolly the sheep was the first mammal cloned from a somatic cell.

Aging and Health of Cloned Animals

  • Dolly's Case: The premature aging of Dolly highlighted possible health implications related to cloning techniques.
      - Telomere Length: Evidence suggests she may have been "genetically older" than her biological age based on telomere lengths.

Recent Advances in Cloning

  • Success Across Species: Cloning techniques applicable to multiple mammalian species, producing several successful instances of cloned animals.

  • Application in Agriculture: Cloning could yield genetically homogeneous herds, enhancing agricultural productivity.

Overview of Stem Cells

  • Function: Stem cells are critical for developing organisms and replenishing damaged cells in adulthood.

  • Characteristics: They have the capacity to divide and differentiate into specialized cell types.

Types of Stem Cells

  • Totipotent: Able to differentiate into all cell types, e.g., fertilized egg.

  • Pluripotent: Can become almost any cell type but not a full organism, e.g., embryonic stem cells (ES).

  • Multipotent: Can differentiate into several cell types.

  • Unipotent: Can only differentiate into one specific cell type.

Specific Stem Cell Types in Mammals

  • Embryonic Stem Cells (ES cells): Found in the inner cell mass of the blastocyst, pluripotent in nature.

  • Embryonic Germ Cells (EG cells): Germ-line cells located in the gonads during fetal development, also pluripotent.

Stem Cell Applications in Medicine

  • Therapeutic Potential: Stem cells may assist in understanding genetic mechanisms of development and treat diseases or injuries.
      - Examples: Bone marrow transplants help patients with cancer-related blood disorders.

Challenges in Stem Cell Availability

  • Rarity of Adult Stem Cells: Adult stem cells (e.g., in bone marrow) are scarce (1 in 10,000).

  • Ethical Dilemmas: The sourcing of embryonic stem cells from unused embryos or aborted fetuses raises ethical concerns.

Induced Pluripotent Stem Cells (iPS)

  • Discovery: In 2006, techniques were developed to reprogram adult mouse fibroblasts into pluripotent stem cells by introducing four transcription factor genes.

  • Potential: iPS cells differentiate into various cell types and offer ethical alternatives to traditional embryonic stem cells.