(4)Transgenic Animals Notes

Transgenic Animals

Introduction

• Selective breeding enhances traits but requires generations.

• Introducing new traits into established lines is difficult.

• SCNT allows gene insertion into higher organisms.

• Cloned genes are inserted into fertilized eggs, implanted, and bred to create transgenic offspring.

Terminology

• Transgenic: animal with altered genetic composition via foreign DNA.

• Transgene: introduced DNA.

• Transgenesis: the process.

• Pharming: obtaining drugs from transgenic animal milk.

Transgenic Mice

• Mice are a common research model for gene regulation, tumor development, and immunology.

• Used to assess human therapeutic drug production feasibility.

Transgenesis Methods:

• Retroviral vector method.

• DNA microinjection method.

• Engineered embryonic stem cell method.

Retroviral Method

• Effective for small DNA transfer.

• Rarely used commercially due to potential retroviral genome integration.

• 8-cell stage embryos are infected with defective retrovirus, resulting in transgenic pups.

DNA Microinjection Method

• Donor females are superovulated using hormone injections.

• Fertilized eggs are collected, and transgenes are microinjected.

• Eggs are implanted into foster mothers.

Identification

• Southern blot hybridization.

• Mating transgenic mice to create pure lines.

Efficiency

• 66% egg survival post-injection.

• 25% implanted eggs develop into pups.

• 25% of pups are transgenic.

Engineered Embryonic Stem Cell Method

• ES cells from blastocysts can differentiate into any cell type.

• ES cells can be genetically engineered to create transgenic animals.

• Avoids issues of microinjection and retroviral methods.

• Cells with integrated DNA at target sites are enriched.

Positive-Negative Selection Method

• Uses homologous DNA blocks (HB1, HB2), a transgene, G-418 resistance, and thymidine kinase genes (tk1, tk2).

• Ganciclovir kills cells expressing thymidine kinase.

Targeting DNA Vector

• Contains homologous DNA blocks and cloned bacterial DNA.

• Primers (P1, P2) detect site-specific integration.

• Random integration will not synthesize the predicted amplified DNA product.

Knockout

• Involves inserting a selectable marker gene within a functional gene.

• Used to study gene inactivation effects and model human genetic diseases.

Genetic Modification with Cre-IoxP Recombination System

• Regulates gene expression within a cell type using bacteriophage P1 elements.

• Cre gene cleaves and recombines DNA at loxP sites.

• loxP sites flank an exon; Cre recombinase excises and circularizes the loxP site, inactivating the gene.

• Used to activate genes in specific tissues by excising transcription-blocking sequences.

Genome Editing Using CRISPR-Cas

• Cas9 mRNA and sgRNA are microinjected into fertilized eggs.

• Can cause chromosomal deletion, transgene insertion, short sequence insertion or chromosomal translocation.

Conditional Regulation of Gene Expression

• Transgenes can be turned on/off using the tetracycline-inducible system.

• Doxycycline (Dox) turns off transgene expression in the tet-off system.

• tet-off system involves a hybrid protein (tTA) that binds to tetO promoter sequences.

• In the presence of Dox, a Dox-tTA complex is formed.

• The tet-on system requires Dox for transgene transcription via a mutated tet repressor.

• Used in mouse models for Huntington's disease (HD).

Huntington's Disease Model

• HD involves an increased CAG unit in the HD gene, leading to glutamine repeats.

• Mouse models use a transgene with exon1 and 94 CAG repeats with the tet-off system.

• Dox is removed at birth which leads to the transgenic mice developing HD-like conditions.

Conditional Control of Cell Death

• Inducing cell death aids in studying organ failure and tissue recovery.

• Transgenic mice express human heparin-binding epidermal growth factor receptor (HB-EGFr) under a liver-specific promoter.

• HB-EGFr binds diphtheria toxin, inactivating polypeptide elongation factor 2 (EF-2), causing cell death.

Cloning of Livestock

• Dolly the sheep was cloned via nuclear transfer from an adult mammary cell.

• Demonstrated pluripotency of the nucleus.

• Somatic cell nuclei used to clone cattle, goats, and pigs.

• All offspring are transgenic.

• Transgenic line establishment does not require multiple generations.

Transgenic Cattle, Sheep, Goats, and Pigs

• Dairy cattle are suitable for transgenesis due to high milk production.

• Modified microinjection protocols are used however yield is poor.

Goals of Transgenesis

• Changing milk composition (e.g., increasing k-casein for cheese production, decreasing lactase).

• Developing mammary glands as bioreactors for pharmaceutical protein production.

• Addressing environmental concerns related to phosphorus levels in pig fecal material.

• Reducing hyperacute rejection in animal-human transplants by introducing protective proteins into donor animals.

Transgenic Birds

• Several sperms penetrate the ovum, therefore injected DNA does not integrate into genomic DNA.

• Transgene is injected into the germinal disc region on the yolk.

• Eggs are cultured in vitro, the embryo is then placed in a surrogate egg.

• Pluripotent cells are preferred vehicle for transgenesis.

Transgenic Fish

• Microinjection and electroporation generate transgenic fish for enhanced growth and disease resistance.

• DNA microinjection is done on embryos which reached 4-cell stage development because pronuclie are small.

• Transgenic fish are generated in temperature regulated holding tanks.

Applications

• Growth hormone transgenes to enhance transcription in cold waters (e.g., salmon).

• Biosensors for aquatic pollutants via estrogen-responsive promoters driving GFP expression.

Comparison of Transgenic Mice Generation Methods

1. Retroviral Vector Method

   • Description: Infects 8-cell stage embryos with defective retrovirus.

   • Advantages: Effective for transferring small DNA fragments.

   • Disadvantages: Rarely used commercially due to potential retroviral genome integration.

2. DNA Microinjection Method

   • Description: Transgenes are directly microinjected into fertilized eggs, which are then implanted into foster mothers.

   • Advantages: Simple and direct.

   • Disadvantages: Lower efficiency; requires superovulation of donor females.

3. Engineered Embryonic Stem Cell Method

   • Description: ES cells from blastocysts are genetically engineered to create transgenic animals.

   • Advantages: Avoids issues of microinjection and retroviral methods; allows for targeted integration.

   • Disadvantages: Requires ES cell manipulation.

Determining Site-Specific Integration of a Transgene Cassette

1. Ganciclovir Screening

   • Method: Used in positive-negative selection. Cells expressing thymidine kinase (tk1, tk2) are killed by ganciclovir.

   • Application: Selects against cells with random integration of the targeting vector.

2. PCR

   • Method: Primers (e.g., P1, P2) are designed to detect site-specific integration. PCR amplification confirms the integration at the target site.

   • Application: Random integration will not synthesize the predicted amplified DNA product.

Generating a Knock-Out Mouse

• Homologous Recombination: A selectable marker gene is inserted within a functional gene to disrupt its function.

  • Involves inserting a selectable marker gene within a functional gene.

    • Used to study gene inactivation effects and model human genetic diseases.

• Cre-loxP Recombination: LoxP sites flank the target gene; Cre recombinase excises the gene, inactivating it.

  • Cre gene cleaves and recombines DNA at loxP sites.

  • loxP sites flank an exon; Cre recombinase excises and circularizes the loxP site, inactivating the gene.

Activating Gene Expression Using Cre-loxP Recombination

• In the engineered embryonic stem cell model, loxP sites flank a transcription-blocking sequence.

• Cre recombinase excises this sequence, allowing gene expression.

  • Used to activate genes in specific tissues by excising transcription-blocking sequences.

Gene Knockdown and Conditional Knockdown Using shRNA

• Gene Knockdown: shRNA (short hairpin RNA) is introduced to target mRNA, leading to its degradation and reduced gene expression.

• Conditional Knockdown: shRNA expression is regulated by an inducible promoter, allowing knockdown only under specific conditions.

CRISPR-Cas9 for Genetic Modifications

• Cas9 mRNA and sgRNA are microinjected into fertilized eggs.

  • Can cause chromosomal deletion, transgene insertion, short sequence insertion or chromosomal translocation.

• Types of Modifications: Chromosomal deletion, transgene insertion, short sequence insertion, chromosomal translocation.

Conditional Regulation of Gene Expression Using Tet On – Tet Off

• Tet-Off System: Involves a hybrid protein (tTA) that binds to tetO promoter sequences to activate transcription. Doxycycline (Dox) binds to tTA, preventing its binding to tetO, thus turning off transgene expression.

  • Transgenes can be turned on/off using the tetracycline-inducible system.

  • Doxycycline (Dox) turns off transgene expression in the tet-off system.

  • tet-off system involves a hybrid protein (tTA) that binds to tetO promoter sequences.

  • In the presence of Dox, a Dox-tTA complex is formed.

• Tet-On System: Requires Dox for transgene transcription via a mutated tet repressor.

  • the tet-on system requires Dox for transgene transcription via a mutated tet repressor.

• Huntington's Disease Model: Mouse models use a transgene with exon1 and 94 CAG repeats with the tet-off system. Removing Dox at birth leads to HD-like conditions.

  • HD involves an increased CAG unit in the HD gene, leading to glutamine repeats.

  • Mouse models use a transgene with exon1 and 94 CAG repeats with the tet-off system.

  • Dox is removed at birth which leads to the transgenic mice developing HD-like conditions.

Transgenic Cell Line Susceptible to Liver Cell Death

• Transgenic mice express human heparin-binding epidermal growth factor receptor (HB-EGFr) under a liver-specific promoter.

  • inducing cell death aids in studying organ failure and tissue recovery.

  • Transgenic mice express human heparin-binding epidermal growth factor receptor (HB-EGFr) under a liver-specific promoter.

• HB-EGFr binds diphtheria toxin, inactivating polypeptide elongation factor 2 (EF-2), causing cell death.

  • HB-EGFr binds diphtheria toxin, inactivating polypeptide elongation factor 2 (EF-2), causing cell death.

Transgenic Livestock Generation

• Mechanism: Somatic cell nuclei are used to clone cattle, goats, and pigs.

  • Dolly the sheep was cloned via nuclear transfer from an adult mammary cell.

  • Demonstrated pluripotency of the nucleus.

  • Somatic cell nuclei used to clone cattle, goats, and pigs.

  • All offspring are transgenic.

  • Transgenic line establishment does not require multiple generations.

• Desirability: -Enhanced milk production (dairy cattle), -modified milk composition, -pharmaceutical protein production in mammary glands.

  • addressing environmental concerns related to phosphorus levels in pig fecal material reducing hyperacute rejection in animal-human transplants by introducing protective proteins into donor animals.

  • Dairy cattle are suitable for transgenesis due to high milk production.

  • Modified microinjection protocols are used however yield is poor.

  • Changing milk composition (e.g., increasing k-casein for cheese production, decreasing lactase).

  • Developing mammary glands as bioreactors for pharmaceutical protein production.

  • Addressing environmental concerns related to phosphorus levels in pig fecal material.

  • Reducing hyperacute rejection in animal-human transplants by introducing protective proteins into donor animals.

Transgenic Birds Generation

• Mechanism: Transgene is injected into the germinal disc region on the yolk. Eggs are cultured in vitro, and the embryo is then placed in a surrogate egg.

  • Several sperms penetrate the ovum, therefore injected DNA does not integrate into genomic DNA.

  • Transgene is injected into the germinal disc region on the yolk.

  • Eggs are cultured in vitro, the embryo is then placed in a surrogate egg.

  • Pluripotent cells are preferred vehicle for transgenesis.

Transgenic Fish Generation

• Mechanism: Microinjection and electroporation are used to generate transgenic fish.

  • Microinjection and electroporation generate transgenic fish for enhanced growth and disease resistance.

  • DNA microinjection is done on embryos which reached 4-cell stage development because pronuclie are small.

  • Transgenic fish are generated in temperature regulated holding tanks.

• Applications:
  -Growth hormone transgenes enhance transcription in cold waters (e.g., salmon).
  -Biosensors for aquatic pollutants

Here are the definitions for the requested terms:

Transgene: An artificially introduced gene into an organism's genome.

Transgenesis: The process of creating a transgenic organism by inserting foreign DNA.

Pharming: The production of pharmaceutical products in transgenic animals, often through their milk or eggs.

Embryonic Stem Cell (ES Cell): A pluripotent cell derived from the inner cell mass of a blastocyst, capable of differentiating into any cell type, used in creating transgenic animals.

Gene Knockdown: Reducing the expression of a specific gene, typically by introducing short hairpin RNA (shRNA) to target and degrade mRNA.

Conditional Gene Knockdown: Gene knockdown that is regulated by an inducible promoter, allowing the reduction of gene expression only under specific conditions.

Gene Knockout: A genetic technique in which a specific gene is inactivated or disrupted, often by inserting a selectable marker gene into the functional gene.

Gene Conditional Knockout: A knockout where a gene is inactivated in a specific tissue or at a specific time, often using Cre-loxP recombination to excise the gene.

Tet-Off System: A system where gene expression is active until tetracycline or doxycycline (Dox) is added; Dox binds to tTA, preventing its binding to tetO and turning off transgene expression.

Tet-On System:

• Gene expression is turned on in the presence of tetracycline or doxycycline (Dox).

• A mutated tetracycline repressor protein requires D