Biotechnologies CHAPTER 17

Selected Biotechnologies

RNA Interference

  • Natural defense mechanism designed to destroy foreign/harmful RNA.

  • Mechanism can be manipulated to silence a specific gene of interest.

DNA

  • The Central Dogma

    • Illustration of the flow from DNA to RNA to protein including:

    • DNA molecule

      • Gene sequences: Gene 1, Gene 2, Gene 3

      • Sequence example: AA A CCGG C A A A A

    • mRNA production through transcription.

      • mRNA molecules:

        • Example Sequence: @@@AAAA

        • Additional Sequence: MMMGGCCGUMMŬ

    • Process of translation to form proteins.

    • Key components:

      • Transcription

      • mRNA (messenger RNA)

      • Translation

      • Polypeptide formation

      • Codons and amino acids involved in translation.

Anti-Sense RNA

  • Definitions:

    • mRNA is referred to as "sense".

    • Any complementary sequence to mRNA is termed "anti-sense".

  • The formation of a duplex from anti-sense RNA can inhibit translation.

  • Example process flow:

    • Gene Transcription -> Messenger RNA formation -> Antisense RNA -> Duplex formation -> Translation Blocked.

Discovery of RNAi

  • Mechanism evidenced as:

    • Overexpression suppression when extra gene copies (Petunia flower color gene) were used.

    • Resistance observed in tobacco plants exposed to virus genes.

    • In roundworms (C. elegans), the expression suppression by double-stranded RNA is more effective than anti-sense RNA.

Mechanism of RNAi

  • Process overview:

    • Double-stranded RNA (dsRNA) is recognized and cleaved by Dicer into 22 base pair (bp) sequences known as small interfering RNA (siRNA).

    • siRNA is recognized by the RNA-induced silencing complex (RISC).

    • RISC unwinds siRNA, retaining one strand for specific binding to target mRNA.

Activities of RISC

  • Endonuclease Activity:

    • Slicer enzyme cleaves mRNA.

    • Other ribonucleases (RNases) may further degrade mRNA.

  • Amplification of RNAi:

    • siRNA can be amplified, promoting anti-sense RNA extension.

  • Blockage of Translation:

    • RISC directly blocks translation by physically binding to mRNA.

Slicer and Amplification

  • Mechanism Flow:

    • Dicer cleaves dsRNA to form siRNA duplexes which become active siRNA.

    • Target Recognition: RISC aids in identifying specific mRNA.

    • RNA Synthesis: RdRP complex generates secondary siRNAs which can subsequently be cleaved by Dicer.

    • Target Cleavage by RISC: RISC facilitates the cleavage of target mRNA.

Applications of RNAi

  • Research Applications:

    • Gene function studies can be conducted by blocking gene expression, particularly useful for lethal genes.

    • Ability to silence multiple genes simultaneously, akin to global repression seen in microarrays.

  • Therapeutic Applications:

    • Can prevent viral spread (e.g., HIV, polio, hepatitis C).

    • Stop cancer progression by silencing oncogenic (cancer-causing) genes.

    • Apply to any disorder requiring gene silencing.

Gene Knockouts Using Neo

  • Process Overview:

    • Step 1: Insert neomycin (neo) resistance gene into target gene disrupting its function.

    • Step 2: In certain embryonic stem (ES) cells, the construct recombines with the chromosomal gene (double crossover event).

    • Step 3: ES cells cultured on G418 medium selecting for cells that have the gene disrupted (those carrying neo).

    • Step 4: These ES cells are injected into a blastocyst and implanted into a surrogate mother to develop genetically modified offspring.

    • Step 5: Offspring can be homozygous for the knockout gene, assessing resulting phenotypes ranging from lethality to unaffected.

Stem Cells

  • Differentiation:

    • Once differentiated, animal cells generally cannot revert to a previous state, although new technologies are emerging.

    • Types of Stem Cells:

    • Adult stem cells: have limited differentiation potential.

    • Embryonic stem cells: capable of differentiating into any cell type.

Plant Cloning

  • Totipotency of Plant Cells:

    • Example: Carrot root cell

    • Culturing 2-mg fragments in nutrient medium leads to division and the formation of an embryonic plant from a single cell.

    • The plantlet eventually transfers to soil and develops into a mature carrot plant.

Animal Cloning - Nuclear Transplantation

  • Steps Involved in Cloning:

    1. Donor mammary cells are cultured and provide a nucleus.

    2. Egg cells from a donor have their nuclei removed.

    3. Cells are fused (the nucleus from mammary replaced in the egg).

    4. Fused cell is grown in culture to form an early embryo.

    5. The embryo is implanted into a surrogate mother, resulting in a lamb, "Dolly", who is genetically identical to the mammary cell donor.

DNA Modification During Development

  • Methylation:

    • Prevents gene expression in differentiated cells.

  • Key changes occur at:

    • Fertilization

    • Fetal development

    • Postnatal stages.

  • Histone Modification:

    • Examples: Methyl and acetyl group modifications.

Antibodies and Immune Response

  • Antibodies play a critical role in defending against infections caused by foreign molecules (viruses and bacteria).

  • Functionality:

    • Antibodies form aggregates with antigens, which are then ingested by phagocytic cells.

    • Blood proteins can kill bacteria or viruses coated with antibodies.

The Antibody Molecule

  • Antibody structure:

    • Proteins with two identical light chains and two identical heavy chains, possessing two identical antigen-binding sites.

Raising Antibodies

  • Process:

    • Animals are injected with the protein of interest.

    • Subsequent blood draws yield serum containing specific antibodies.

Affinity Chromatography Using Antibodies

  • Immunoaffinity Techniques:

    • Mixtures are introduced to antibodies linked to beads in a chromatography column.

    • The aggregates are collected and purified by centrifugation and elution processes.

Immunohistochemistry and Immunoassays

  • Methods for visualizing or detecting specific molecules or cell components.

  • Chemicals such as peroxidase or alkaline phosphatase interact with substrates to yield colored products for detection.

Environmental Applications

  • Biofuels: Production through biochemical processes utilizing algae growth and harvesting.

  • Waste Cleanup: Techniques that can detoxify and render waste safe.

  • Sunlight Utilization: Integrating gas and water conditioning into biomass processing.

Crop Improvement with Agrobacterium tumefaciens

  • Utilizes Ti plasmids for introducing genes into plant genomes, allowing for new traits in crops.

Therapeutic Cloning

  • Overview:

    • Nucleus from a patient’s skin cell (e.g., diabetic patient) is transplanted into a denucleated egg cell.

    • Following cleavage, the embryonic stem cells (ES cells) generated can develop into specific cell types, such as pancreatic islet cells, which are transplanted back to benefit the patient.

Ethics of Biotechnology Use

  • Key Considerations:

    • Genetically Modified Products: Safety concerns related to GM foods and environmental impact; ownership rights in intellectual property.

    • Screening & Privacy Issues: The potential for discrimination based on genetic screening results; variances in public and private sector access to genetic information.

    • Inappropriate Use: Ethical dilemmas surrounding human cloning and risks of bioterrorism.