Transgenic Animals and Molecular Techniques

Overview/History

• Transgenics: Gene transfer into intact organisms (Gordon and Ruddle, 1981).
  • Includes adding new genes, modifying existing genes, and swapping whole genomes.
• Milestones:
  • Jaenisch (1976): Novel gene inserted into mouse embryos via viral infection.
  • Gordon et al. (1980): Pronuclear injection of DNA into mouse embryo greatly increases efficiency.
  • Palmiter et al. (1982): Creation of giant mice using pronuclear injection of growth hormone transgene.
  • McGrath et al. (1983): Technique for pronuclear transplantation in mammals devised.
  • Hammer et al. (1985): Creation of transgenic livestock by pronuclear injection.
  • Clark et al. (1989): First commercially viable transgenic bioreactor producing human Factor IX.
  • Campbell et al. (1996): Cloning from manipulated cultured cells opens the way for generating targeted mutations in livestock.
  • Wilmut et al. (1997): Clone of an adult organism generated: “Dolly”.

Learning Outcomes

• List the advantages and disadvantages of recombinant protein production in animals versus other commonly-used systems such as bacteria.
• Give examples of transgenic animals and what they are used for.
• Describe the molecular tools (recombinant DNA) needed to make a transgenic animal.
• Indicate the embryological manipulations involved in order to get recombinant DNA into an animal.

Further Reading

• Essential Cell Biology by Alberts et al. (pp. 340-342).
• Principles of Development by Wolpert (pp. 108-109).
• Developmental Biology by Gilbert (pp. 69-72).
• Molecular Cell Biology by Lodish et al. (pp. 281-289).
• Larrick and Thomas, Curr. Opinions in Biotech. 12:411 (2001).
• Transgenic animals, Houdebine (ed).
• Nature Biotechnology (any issue).

Transgenic Animals Examples:

• Belgian Blue cattle: Example of extreme muscle growth, though the transcript asks whether they are the result of transgenics. (Linde)
• Callipyge ram ("Solid Gold"): Mutation leads to muscle overgrowth in hindquarters (Georges et al., Trends in Genetics 19:248 (03)).
• Tracy the Transgenic Sheep: Produced human alpha-1 antitrypsin (AAT) in her milk to treat emphysema.

Tracy the Transgenic Sheep: AAT Production

• Emphysema: Lung degeneration caused by a genetic mutation, leading to deficiency in AAT.
  • AAT protects lung tissue from enzymes like trypsin and elastase.
• Current treatment: Inhaling AAT aerosol spray.
  • Requirement: 200g AAT per year per patient.
  • Source: Extracted from blood donations in small quantities.
• AAT gene cloned but cannot be produced in bacteria because AAT is a glycoprotein
• AAT now produced by genetically-modified sheep (e.g., Tracy).

Requirements of Transgenic Animals for Pharmaceutical Production

• High levels of desired protein production without endangering the animal's health.
• Ability to pass on protein production to offspring.
• Combine DNA for protein synthesis with DNA that directs the protein to the mammary gland.

Tracy: The Most Valuable Sheep

• 1990: PPL Therapeutics transferred human AAT gene into five sheep embryos.
• Tracy produces 35 grams AAT per liter of milk.
• Milk collected for the sheep's lifetime; proteins separated using column chromatography (30% AAT recovery).
• Genetically engineered AAT similar to natural human AAT in laboratory studies.
• Clinical trials are ongoing.
• Estimated value: $7,000 per liter of milk.

Techniques to Generate Transgenic Animals

• Basic manipulation of embryos.
• Retroviral infection.
• Pronuclear microinjection.
• Stem cell techniques.
• Nuclear transfer.

A. Basic Manipulation of Embryos

• In vitro fertilization (IVF) techniques (Artificial Insemination (AI) and twinning) used commercially to increase reproduction of valuable animals.
  1. Collection of eggs:
     • Stimulating greater numbers of eggs to release than normal (superovulation) by giving hormone injections
     • Collection of the unfertilized eggs
     • Subsequent in vitro maturation (IVM) of the eggs if necessary
  2. Collection of sperm
     • Postmortem or using artificial insemination (AI) techniques.
     • In vitro capacitation (“priming”).
     • Selection of “good” sperm.
  3. In vitro fertilisation
     • Combining harvested eggs and sperm in the lab
     • Fertilized eggs start to divide after a few hours
     • Embryos can be grown in special nutrient solutions under an atmosphere enriched in CO_2 and humidified (an incubator)
     • Embryos will progress to the stage at which they would normally implant in the womb or uterus in vitro
     • At this point the embryos need to be transferred to a foster female
     • The foster is stimulated to receive the embryos by mating to a vasectomised male
  4. Twinning: Carried out on embryos at early stages (2-16 cell approx.).
     • Removing the “shell” (zona pellucida) and applying pressure splits the mass of cells in two
     • Each half then goes on to form a complete embryo
  5. Aggregation chimeras: Two embryos without the ZP can be made to fuse together by placing in a confined space
     • Leads to a mosaic animal

B. Use of Viruses to Generate Transgenic Animals

• First technique used, recent revival of interest.
• Viruses integrate DNA into host cell DNA.
• Virus DNA is modified to be less harmful, gene of interest added.
• Hampered by mosaicism, size restrictions, and transgene expression.
  1. Modification of virus to make non-harmful
  2. Addition of gene of interest
  3. Infection of embryos
  4. Transfer of surviving embryos to a foster female
  5. Birth of offspring and screening for presence of gene
• Involves putting the gene of interest in a virus and exposing embryos to the virus. Subsequent infection results in uptake of the viral DNA into the embryo’s genome and can result in expression of the transgene.

C. Pronuclear Microinjection

• Most widely used technique for generating animals with new genes, may be superseded by nuclear transfer.
• Requires basic embryological techniques and recombinant DNA technology.
• Basic idea: add a new gene into the target animal.
• Alternative techniques: SCNT, iPS, TALEN, CRISPR, ZFN.
• Stages in a typical experiment:
  1. Putting gene in a form suitable for injection
  2. Collection of one-cell embryos from target animal
  3. Injection of gene into embryos
  4. Growth and selection of viable embryos in vitro
  5. Transfer of surviving embryos to a foster female
  6. Birth of offspring and screening for presence of gene
• Fertilization of the egg in mammals
  • The sperm and egg nuclei have half the normal number of chromosomes and are called pronuclei.
  • The sperm pronucleus travels across the egg and fuses with the female pronucleus, restoring the normal number of chromosomes
  • Can inject DNA at these stages

Molecular Tools

• Need to get an animal to produce a new protein.
  • Injecting protein is not effective as they are quickly broken down
• The gene for the protein contains needed information.
  • Gene: Stretch of DNA coding for one protein.
• DNA is a stable form of information and not broken down
• DNA is similar in all organisms; instructions in DNA from one species can be read by cells from another species.
• Introducing new DNA into a cell leads to stable reproduction along with the cell’s own DNA.

Expression Construct

• Includes a promoter (e.g., Beta-lactoglobulin for mammary gland expression) and a gene of interest (e.g., Factor IX).
• A simple expression construct:
  • Gene A gene of interest eg the gene for Factor IX protein is cloned into a plasmid together with a stretch of DNA which tells the cell to turn on the gene only in the mammary glands (the promoter). Together, these two pieces of DNA are called an expression construct
• Comparison between a procaryotic and eucaryotic gene
• Genes from mammals and other higher organisms are interrupted by stretches of DNA which do not form part of the protein (introns). These are cut out by the cell before the protein is made.

Tracy the Transgenic Sheep - The Making Of:

1. Female sheep stimulated with fertility drugs to increase egg production and collection from ovaries
2. In vitro fertilization of eggs
3. Plasmid preparation containing human AAT gene and Beta-lactoglobulin promoter sequence. AATB construct is appoximatley 4.0 kb of the 5’ end of the ovine BLG clone SS1 fused to a minigene encoding human AAT.
4. Microinjection of hundreds of copies of the plasmid into the male pronucleus of fertilized zygotes
5. Zygotes divide in vitro until 16-cell stage
6. Implant 16-cell embryos into surrogate mother ewes. Low implantation rate results in successful pregnancy.
7. Test offspring for AAT production in thier milk

The Making of Tracy the Transgenic Sheep

• Step 1: Female sheep given fertility drug to stimulate egg production, mature eggs collected from ovaries.
• Step 2: Eggs fertilized in vitro.
• Step 3: Plasmid prepared containing gene for human AAT and promoter sequence for β-lactoglobulin.
  • The AATB construct comprises ~4.0 kb of the 5’ end of the ovine BLG clone SS1 fused to a minigene encoding human AAT. Thick line 5’ BLG sequences; Open box, BLG exon 1 sequences; yellow boxes AAT exons; thin lines AAT introns and 3’ flanking regions. Start, stop codons and polyadenylyation site shown.
• Step 4: Zygotes divide in vitro until the 16-cell stage.
• Step 5: 16-cell embryos implanted into uterus of surrogate mother ewes; few implantations result in successful pregnancy.
• Step 6: Offspring tested for AAT production in milk to find if the zygote took up AAT gene for its expression. Less than 1% success rate.

Common Problems in Transgenic Animal Generation

• Chimerism: DNA is not taken up at once in the one-cell embryo, but integrates at the 2 or 4 cell stage. It is not present in every cell, resulting in a chimeric animal. This animal may not produce the transgene in the target organ or it may not produce transgenic offspring
• Silencing: Transgene produces protein at very low levels, despite no apparent problem with the gene due to the transgene inserting in an inactive area of the genome
• Low Yields: In typical attempts to create transgenic animals by pronuclear microinjection, less than 5% of animals integrate the expression construct and only a fraction of these express sufficient quantities of the protein to be of any commercial interest.

Summary

• Transgenic animals generated using pronuclear injection.
• DNA solution injected into embryos at one-cell stage; some embryos incorporate DNA into nucleus.
• Transgene present in every cell of transgenic animal and passed to progeny.
• Transgene contains gene for protein of interest and promoter driving expression in the target organ (expression construct).
• Transgenic animals are generated at low efficiency, but can have great advantages once made.