In some cases overexpression of human genes in bacteria (such as E. coli) does not yield a protein that is functionally active in humans. The reason for this is that some proteins need to be post-translationally modified (phosphorylated, glycosylated, etc.) before they are active. Bacteria generally lack the specific enzymes recognizing the human protein sequences that need to be modified, and thus the bacterially produced gene product will differ from the native one. To counter this problem, certain human genes can be introduced into farm animals (usually yeast will do the job, too), and when these genes are expressed in the mammary glands of the animals, the post-translationally modified protein can be isolated from milk, tested whether its post-translationally modified product is identical or at least very similar to the native human one, and if so, be developed as a pharmaceutical. For example, the genes for two different human blood clotting factors (VIII and IX) have been hooked up to sheep and pig regulatory sequences that causes expression in mammary tissue; after transformation of sheep or pig embryos, genetically engineered animals have been selected that produce milk with a large percentage of human blood-clotting factor. This protein can be isolated from the milk, purified, and marketed. Similarly, transgenic rabbits have been created that produce human interleukin-2, which is a protein stimulating the proliferation of T-lymphocytes; the latter play an important role in fighting selected cancers.
Other human proteins that have been expressed in transgenic animals include: anti-thrombin III (to treat intravascular coagulation), collagen (to treat burns and bone fractures), fibrinogen (used for burns and after surgery), human fertility hormones, human hemoglobin, human serum albumin (for surgery, trauma, and burns), lactoferrin (found in mother milk), tissue plasminogen activator, and particular monoclonal antibodies (including one that is effective against a particular colon cancer). Animals mostly used for this work are pigs, cows, sheep, and goats.
The amounts of milk needed to provide a national supply of these pharmaceuticals are really very reasonable. Assuming the animals produce 1 g of the protein per liter milk and one has a purification efficiency of 30% (that is, 30% of the protein is recovered in the pure sample), then a pig can produce 75 g of protein per year, a goat 100 g, a sheep 125 g, and a cow 3 kg. As the national need of blood-clotting factor IX is 2 kg / yr, one cow per country can do the job. For other proteins the demand is larger (for example, for tissue plasminogen activator it is 75 kg per year and for human serum albumin it is about 1,000 kg / yr), but nonetheless a limited number of animals is all one now needs to meet the national demand for pharmaceutical proteins that used to be astronomically expensive.
Dolly and Polly
A fairly large stir was caused in the popular media when a group in Scotland associated with the Roslin Institute and with PPL Therapeutics announced in early 1997 that a lamb, Dolly, had been born that had been cloned from a single cell taken from her mother's udder. Of course, cell division of vegetative cells to eventually yield a new organism with the same genetic makeup as the parent is pretty usual among "lower" organisms and certain plants, but until 1997 it had never been shown for mammals. The creators of Dolly had taken an unfertilized egg cell with the nucleus removed, and fused that with the cell from the udder. The fused cell was made to divide and developed into a normal embryo. This was implanted into a surrogate mother, and it developed into a healthy lamb. This was the first time that genetic information from a fully differentiated, vegetative mammalian cell was used to give rise to a new, fully differentiated organism.
Building upon the "success" of Dolly, the next step came the same year from the same group in Scotland. The single, diploid cell originating from the adult sheep now was genetically altered (introducing a human gene gene coding for blood clotting factor IX) before fusing with a denucleated egg cell. The fused cell was made to divide and to develop into an embryo, which was implanted into a surrogate mother. The resulting lamb, Polly, contains the human gene in every cell of her body.
The method resulting in Polly is seen as a major improvement as compared to the technology of the early nineties that led to the first transgenic bovine creature, Herman, carrying the human lactoferrin gene (lactoferrin is an important component in breast milk). At that time, genes were injected into newly fertilized eggs, and only in rather infrequent cases did the gene stably integrate and lead to a transformed animal.
Now that Dolly is an adult, questions are being raised regarding her health. In most respects she is a normal sheep. She is fertile and has given birth to a number of lambs. However, at a young age she developed arthritis, which usually does not occur in sheep until much later. Whether or not this is a consequence of cloning (which may not set back the biological clock) is as yet unknown. Her telomers are a little shorter than usual for an animal of her age, but whether this has an impact on her health and longevity is unclear.Dolly in most respects was a normal sheep. She was fertile and has given birth to a number of lambs. However, at a young age she developed arthritis, which usually does not occur in sheep until much later, and she died at 6 years of age (much younger than the average lifetime of sheep) due to a progressive lung disease. Whether or not this is a consequence of cloning (which may not set back the biological clock) is as yet unknown. Her telomers were shorter than usual for an animal of her age, but whether this had an impact on her health and longevity is still unclear.
In some countries transgenic fish has been developed. This is quite easy, as there is usually no problem to get female gametes: just squeeze the female; no surgery, no microscopes. The egg cells can be just electroporated to introduce the desired DNA. However, one does not need "high tech" approaches to increase aquaculture yields. Often it is sufficient to have fish male and female hormones produced in bacteria, and utilize these hormones. For some fish, after hatching a male fingerling exposed to estrogen will become female in appearance, while remaining genetically male. The "pseudofemale" can lay eggs producing viable offspring, in spite of the male chromosomes. Female fingerlings can be sex-reversed in the same way through exposure to testosterone, becoming reproductively viable pseudomales.
This application is attractive to fish farmers, who often want all-male groups of fingerlings: they grow faster than females and single-sex ponds mean that a second generation of fingerlings ("recruits") is not produced when the stocked fish becomes sexually mature. Recruits will eat, but will not be marketable by harvest time, thus bringing down production.
However, understandably consumers are not very enthused about having fish that has been exposed to hormones. To avoid having to hormone-treat fingerlings that will be harvested later, one can use a male parent with 2Y chromosomes and no X. All progeny will be XY and male, without needing any hormone treatment. YY males are indistinguishable from XY males and can be obtained from a normal male (XY) and a pseudofemale (a male that was treated with estrogen). Half of the resulting progeny is normal XY male, 1/4 is female (XX) and 1/4 is YY ("supermale"). XYs and YYs can be distinguished from each other by DNA typing. If one wants to produce progeny that is 100% female, one can simply cross a "pseudomale" (XX) with a normal female.
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Center for Bioenergy & Photosynthesis
Arizona State University
Room PSD 209
Tempe, AZ 85287-1604
13 February 2006
phone: (480) 965-1963
fax: (480) 965-2747