In earlier lectures I described how transgenic/transformed bacteria, yeast and plants have been produced. With this in mind, it is fairly straightforward to design methods to obtain transgenic animals such as mice or sheep. Many of the same principles apply, including the barriers that the DNA must cross, whether or not the DNA integrates into a chromosome, and using selectable markers to identify transformed cells. However, there are a number of significant and important differences to consider when producing transgenic animals.
First, we will consider transformation of individual cells that are growing in tissue culture. Many different types of cells can be isolated from animals and grown in sterile culture, normally in a liquid medium in Petri dishes. These cell lines in culture are widely used for many different experiments, examining the cellular and molecular biology of animal cells.
There are a number of physical methods to introduce DNA into animal cells growing in culture. One of the uses of this type of experiment was to identify the functions of specific pieces of DNA. The methods used for transformation of this type of cells include:
With each of these methods, the DNA that is used for transformation is typically a plasmid that contains the gene of interest that you wish to transfer into these cells, frequently accompanied by a selectable marker gene which will allow the transformed cells to grow in the presence of an inhibitor such as an antibiotic.
Overheads to describe these 3 methods.
Electroporation of animal cells is very similar to the procedure used for plant cells. The only significant difference is that animal cells do not contain a rigid cell wall, and so preparation of protoplasts (removal of the cell wall) is not required. Animal cells and DNA are mixed together and then subjected to an electric shock. It is believed that the electric shock produces small temporary pores in the membrane that allow DNA to enter the cell. At least some of the DNA can travel to the nucleus.
Liposomes are artificial membranes that can be formed in a test tube. DNA can be mixed with the liposome preparation under the appropriate conditions. This results in the encapsulation of DNA into synthetic lipid membranes. When this membrane fuses with the cell plasmamembrane, DNA is released into the cell and somehow ends up in the nucleus.
Calcium phosphate precipitates of DNA form when DNA is mixed with calcium chloride. When these DNA precipitates are added to animal cells growing in culture, the precipitated DNA can be taken up by the cells, again transferred to the nucleus and expressed.
Each of these methods allows delivery of DNA to the nucleus of these transformed cells. In many cases, these transformation experiments are carried out to look at the functions of genes in short term studies. In these experiments, there is no need to obtain stably transformed cells and so no selection is required. If stably transformed cells are needed, the cells are subjected to selection so that only transformed cells will grow. If selection is maintained, some of the plasmid DNA molecules will integrate a chromosome at random sites.
It is also possible to use biological vectors to carry specific genes into animal cells. These vectors are derivatives of viruses that infect animals. These viral vectors can be divided into 2 groups:
The free replicating viruses cannot produce stable transgenic cells, because the genetic material that is introduced does not become integrated into the host genome. But this is still very useful for transient, not permanent, introduction of a gene. This approach may have applications in human gene therapy. This has not developed as rapidly as predicted by some, but this idea holds the promise of treating some diseases that are caused by genetic defects or modifications.
The integrating viruses such as HIV enter the cell, copy their RNA genome into DNA, and then this DNA integrates into the chromosome of the host. The genes carried on the virus can then be expressed, and also stably inherited after cell division. The integration of these retroviruses into the host genome is one reason why these viruses are so dangerous. Unless all the cells that carry the virus are removed, the host may still be attacked when the virus becomes re-activated. One limitation of the new triple therapy treatments for HIV is that these drugs do not remove the virus that has integrated into some cells.
With either type of virus vector, not all of the cells in the animal host, are transformed. The progeny of the infected animal will likely not be transformed. Why not? Because the cells that give rise to gametes, eggs or sperm, have not been transformed. Heritable transformation requires that the germ line cells are transformed.
There are a number of methods used to produce transgenic animals, and I will describe two of these methods. Unlike plants, it is extremely difficult to regenerate a fully differentiated animal from a single transformed cell growing in culture. I used to say it was impossible, but the arrival of Dolly, the cloned sheep, now makes it just extremely difficult but not impossible.
The first method is injection of DNA directly into the nucleus of fertilized eggs.
Overhead of process of injection, and pictures.
Mouse eggs are either fertilized in a Petri dish, or fertilized eggs are removed from an impregnated female mouse. Individual eggs are held stationary with a suction pipet. A few picoliters (10-12 of a liter) of DNA solution (about 1,000 molecules of DNA) are injected into one of the nuclei. The eggs are then implanted into a pseudopregnant foster mother. Baby mice are born and tested for the presence of the DNA that was inserted. This can be done using a variety of methods including PCR and DNA blot analysis.
This method of transformation is technically complex and efficiencies are not high. In mice, >90% of the eggs survive injection of DNA, ~20% of these are able to develop to full term upon reimplantation, and ~25% of these may be truly transgenic. In other mammals (e.g. sheep, pigs, cattle) efficiencies are even lower than in mice. However, over time these techniques are becoming more routine and efficiencies are increasing.
The DNA that is inserted into these mammalian genomes is typically:
This leads to some of the same problems of "position effect" variation that I described in plant transformation, where each individual transgenic animal must be regarded as a unique, and the level of expression of the inserted gene varies from animal to animal.
The second method for producing transgenic animals involves transfer of transformed embryonic stem cells into embryos. Embryonic stem (ES) cells are the closest thing to totipotent cells that can be obtained from a mammal. ES cells are isolated from young developing embryos. These cells are grown in culture to establish a cell line. Under appropriate conditions, ES cells can develop into a variety of differentiated cell types. However, for transformation, ES cells are injected into a developing embryo where they give rise to a chimeric embryo. The animal that results from this chimera can be used to establish a line of transgenic mice .
ES cells are transformed by any of the physical methods described above (electroporation, etc.). Transgenic ES cells are identified and propagated. The advantage of this method over simple injection of DNA into a fertilized egg is that very precise changes can be made in the genome of these ES cells, rather than random integration of multiple copies of a gene. For example, it is possible to precisely remove a single gene from the mouse genome, or make a precise change in a specific gene. The transformed ES cells are then injected into young blastocyst embryos. The resulting embryos will be chimeric mixtures of normal and transgenic cells, producing chimeric mice which are normally identified by their mottled appearance because of chimeras comprised of black and white cells. Chimeric mice are bred to identify offspring that have inherited the transgene.
Transformation systems have been developed for many animals, including farm animals such as cows, pigs, sheep and chickens. One long term application of this transformation technology is to improve the quality of farm animals and reduce the cost of production in various ways. Howwever, in contrast to the successes that we have discussed in plant biotechnology, there have been no similar advances made with animals.
Attempts have been made to increase the growth rate of pigs by expressing a chimeric gene to increase growth hormone production in transgenic pigs. While pigs were produced that carried this gene, and they did have increased muscle and reduced fat, these potentially beneficial traits were accompanied by a large number of deleterious abnormalities. It is possible that these negative effects were the result of this growth hormone being expressed in a very uncontrolled manner, in a wide range of tissues. Greater control over the tissues that expressed the growth hormone might eliminate these problems.
A successful example of development of transgenic animals comes from inserting a gene to overexpress growth hormone in salmon. This resulted in a large increase in weight gain in the transgenic salmon. A lot of salmon are raised in aquaculture systems nowadays and the rate and efficiency with which the fish convert the supplied feed into marketable salmon filets is very important to the financial success of these fish farming operations. I understand that this is under further development.
Other traits that have been suggested as targets for biotechnology in production animals include:
One area of animal biotechnology that has attracted a great deal of interest, and shows promise, is the use of animals to produce therapeutic proteins to treat human diseases. The strategy that is being used by many companies is to express these target, high value proteins in the milk of large animals such as pigs, sheep or cows. Milking is a well established procedure, and it should be feasible then to recover and purify the proteins from the milk in large quantities.
To express a new protein in the milk of a transgenic sheep requires that the open reading frame for that protein be used to construct a chimeric gene with the following components:
Transgenic animals are produced that contain this gene. Females are then evaluated to see how much of the new protein is being made in the milk. Those with the highest expression are then selected for further analysis, breeding to develop a herd of producers, and production scale-up. Examples of proteins that are being produced in this way include:
There are a number of advantages of using this approach of production in mammalian milk over microbe production systems. First, it may be possible to reduce production costs. However, the more important reason is that many proteins in eukaryotes are modified in ways that cannot be reproduced in microbes. These modifications can include addition of complex sugars to specific amino acids and cutting the amino acid chain into smaller peptides. Modifications like this are in many cases absolutely critical for the protein to function. These can be made when the protein is expressed in the mammary gland, but not in bacteria. So animal expression systems may have the advantage of producing an active functional protein, compared to what can be made in bacteria.
An excellent article on the topic of using animals for drug production was published in the January 1997 issue of Scientific American, pp.70-74.