Barnyard 101: An Introduction to Transgenic Farm Animals

Commentary by Thomas M. Zinnen

"We have the ability today to probably transform any cell type," said Carl Pinkert in his opening remarks to the Transgenic Animals in Agriculture conference held August 24-28 in Tahoe City, California.

"But it's not just transfer of the gene, it's gene function that's key," added Pinkert, a researcher at the University of Alabama at Birmingham. He illustrated his point with a cartoon of two mice. The first asks, "How do you like my new genes?" The second replies with mores questions: "They're nice, but are they expressed? Are they regulated properly? Are they altered during inheritance?"

Pinkert noted that now nearly 20 years after the first transgenic animal scientists can answer those questions.

Why Transgenic Animals?

Interest in transgenic animals originally fell into two broad areas:

1. Production efficiency of farm animals--the original area of interest among many university researcher.
2. Molecular farming: using livestock to produce medicines, nutraceuticals and tissues for transplant to humans.

"Now most of the money is here (in molecular farming)," said Pinkert, "and it's mostly corporate money."

Adding a Gene by Random Insertion

For basic research, Pinkert pointed out two different lines of approach possible with transgenic cells: gain of function or loss of function. To study gain of function merely requires addition of a new gene.

Pinkert underscored the power of studying animals, not just cells. "Anytime we tried to alter one trait with one gene," he said, "in the end the influences of that gene on other traits was something we couldn't know until we had the animals."

During 1982-83 pioneering research featured transgenic mice given a copy of the gene for human growth hormone. In this case, the added gene inserted randomly in the mouse genome. It did not insert at the mouse gene for growth hormone. The addition of the gene for human growth hormone did not inactivate or "knock-out" the genes for mouse growth hormone.

A picture of two mice side by side, one with the extra gene and the other without, gave "visual impact of what this technology might do," Pinkert pointed out. Comparing the size of the mouse with the extra gene to the control was like comparing a softball to a baseball.

But the control of the growth hormone gene's impact on the endocrine system was not sufficient. This brought home the issue of qualitative versus quantitative control of gene expression. Since then mouse researchers have been wrestling with that next layer of problems: how to control genes in time and tissue, "temporally and spatially"?

Modifying Expression of an Existing Gene by Homologous Recombination

To study loss of function of an existing gene is a bit trickier. It requires a system in which a modified or faulty version of a gene is inserted by homologous recombination. The new version inserts by trading places with the existing gene and "knocks out" the existing version of the gene. In homologous recombination, the inserted gene is not randomly inserted into the genome, but rather it is targeted to insert at the site of the existing homologous gene.

"Long life cycles of farm animals slow genetic analysis," said Pinkert. "That's why researchers use smaller, faster-breeding animals such as mice as model systems to test their ideas and their DNA constructs." Furthermore, the mouse is the only model system that combines homologous recombination with cloning to allow the study of modified genes in development of adult animals. Currently, in livestock homologous recombination is possible only with cells grown in tissue culture. This means a scientist can study the effect of knocked-out genes only on the physiology of the cell. The possible role of the gene in development from embryo to adult cannot be tested without a system of cloning: taking the original cell and growing an adult from it.

In 1997 the cloning of Dolly and engineering of Polly have combined transgenesis and cloning. This combination was an essential step in developing in livestock a system of homologous recombination to modify existing genes. Now researchers are eagerly working on making a system of homologous recombination to further test their ideas with farm animals.

Once such a system is available, animal scientists will be able to ask questions about the roles of genes in development from embryo to adult using livestock, not just mice.

Regulatory Issues Leading to Commercialization

Researchers first produced transgenic farm animals in 1985, yet as of 1997 Pinkert notes no products in the supermarket or at the pharmacy are produced using such animals. A major reason is the lack of a clear route to government approval. "Some (regulatory issues) are still outstanding, affecting utility and acceptance," he said. "Environmental impact is a huge issue" especially for transgenic fish that would likely mingle with their wild relatives.

Commercialization comes with a communication component. "The reasons we put forth for transgenic animals influence public perception," Pinkert warned. He ended by noting that the public perception of pioneering products such as BGH, PST and even FlavrSavr tomato has affected governmental and corporate approach to reviewing and commercializing products from transgenic animals.

Timeline of Animal Cloning and Gene Transfer

• 1891 first successful embryo transfer early 1900's in vitro embryo culture develops
  
  
• 1961 mouse embryo aggregation to produce chimeras
  
  
• 1966 first report of microinjection of mouse embryos
  
  
• 1973 foreign genes function after cell transfection
  
  
• 1974 development of teratocarcinoma cell transfer
  
  
• 1977 mRNA and DNA transferred to Xenopus eggs
  
  
• 1980 mRNA transferred into mammalian ova
  
  
• 1980-81 transgenic mice first documented
  
  
• 1981 transfer of ES cells derived from mouse embryos
  
  
• 1982 transgenic mice and a growth hormone phenotype
  
  
• 1983 tissue specific gene expression in transgenic mice
  
  
• 1985 transgenic domestic animals produced
  
  
• 1985 microinjection for transgenic pigs, sheep, rabbits, fish
  
  
• 1987 chimeric "knock-out" mice described
  
  
• 1987 retrovirus mediated: transgenic chicken
  
  
• 1989 targeted DNA integration & germline chimeric mice
  
  
• 1989 microinjection for transgenic cattle (Russia)
  
  
• 1989 first sperm mediated reports in farm animals
  
  
• 1991 microinjection for transgenic goats first refereed publication
  
  
• 1993 germline chimeric mice produced using co-culture
  
  
• 1996 ES cells used for nuclear transfer: sheep
  
  
• 1997 somatic cells from adult sheep used for cloning by nuclear transfer (Dolly)

Gene Transfer Methods

1. Blastomere/embryo aggregation
   
   
2. Teratocarcinoma cell transfer
   
   
3. Retroviral infection
   
   
4. Microinjection of cells (oocytes) with DNA
   
   
5. Electrofusion
   
   
6. Nuclear transplantation
   
   
7. ES (embryonic stem) cell transfer
   
   
8. Sperm-mediated transfer
   
   
9. Particle bombardment ("gene gun")
   

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