TRANSGENIC MAMMALS ("WILBUR" AS ANOTHER INSTRUMENTAL GOOD)

TRANSGENIC MAMMALS
("WILBUR" AS ANOTHER INSTRUMENTAL GOOD)

Linda de Kort
1992 Woodrow Wilson Biology Institute

Last month the government granted its first patent on something that can look you in the eye. Is this small step for a mouse a giant leap backward or forward for mankind? The New Republic, May 23, l988.

Rationale And Objectives:

This module will provide a real life example of current research using our knowledge of genetic and molecular biology. It will provide an opportunity to examine the ethical considerations of a current development which the students can continue to watch as the school year(s) progress. The students will also be encouraged to explore related issues in other disciplines: patent law issues, status of regulatory procedures, power of stock holders to set policies. They will hopefully begin to understand the complexity and inter- disciplinary nature of an issue.

This module is intended for a second year biology class. Students should have a strong background in the following areas: quaternary structure of proteins, control of gene expression, immune response especially to membrane proteins, blood typing, identification of proteins and DNA through electrophoresis, recombinant DNA techniques. Students also should have previous experience in discussing bioethical problems; Sagoff's " Zuckerman's Dilemma" would serve as excellent background here.

POSSIBLE THOUGH UNTRIED PLAN OF ACTION

Day 1:

Teacher lectures on techniques, history, applications, technical problems.

Teacher assigns article entitled: "DNX Develops Transgenic Pigs" (see References); students are instructed to highlight and prepare outline or concept map by next day class.

Day 2:

Small group discussions. Students are instructed to complete questions on DNX article (see "Technical Questions: DNX"). Hand in one copy/team.

Day 3:

Large group: teacher clarifies and adds to understanding of tech-niques including xenografts. Teacher introduces bioethical con-siderations and gives each group: "DNX-High School Connection" and "Fact Sheet to Accompany DNX Ethical Questions".

Small groups: Begin ethical analysis (see Bill Smith's or Hastings Institute's model), record on melamine boards.

Day 4:

Small group: Complete ethical analysis. Each group also randomly selects a spokesperson to represent their role and another who will play the part of an "unbiased" bioethical committee member.

Large group: hear reports from each group before mock bioethics committee. Committee members will confer and develop policy statement.

Teacher assigns personal bioethical position paper regarding invest-ment in DNX which is due following day. See "Guidelines for Personal Position on DNX Investment".

Day 5:

Teacher collects personal position papers.

Committee spokesperson reports position/policy to class. Dissenting opinions are also encouraged.

Large group: Discuss other ethical considerations not presented. In the "real world" who ought to be making these decisions and how ought they be making them? (See: "Ethical Concerns" and "Issues on Political and Legal Arena".)

TEACHER BACKGROUND INFORMATION

Definitions:

A Transgenic animal is produced in the following manner: gene or genes from another animal are inserted into the fertilized egg or early embryo; this Transgene is later expressed in the organism (the Founder) and passed on to its offspring.

Techniques:

Production of transgene
Two separately cloned genes are spliced together, one gene contains the coding sequence for the protein of interest and the other controls the tissue in which the animal protein will be expressed.
Incorporating gene into parent
There are three methods of generating transgenic mammals.
(a) The genes are usually introduced by micro-injection into the male pronucleus of a fertilized one-cell egg in vitro. This technique is performed by using a capillary pipette whose tip is only several microns wide. These eggs are then injected into a "pseudo-pregnant" (mated with vasectomized male) surrogate female.
(b) Transgenic animals can also be generated from ES cells (embryonic stem cells). ES cells are cells of a very early embryo that still have the ability to differentiate into any tissue. ES cells can be engineered, grown in culture and tested for presence of the gene of interest before insertion into blastocyst. The ES incorporate themselves among the other blastocyst cells; the resulting embryo is a mosaic of original (" wild") cells and engineered cells; these organisms are called chimeras.
(c) Retroviral vectors have also been used to introduce the gene of interest into the stem line; the stem cells are then incorporated into the germ line at the blastocyst stage.
Establishment of Strain
Founder animals can be used to build up a herd with no further need for microinjection. Founder animals are identified by electrophoresis of either blood or milk proteins or genomic DNA using Southern Blot techniques.
Isolation and purification of protein product (in the case of "pharming")
Blood (or milk) flows through a column filled with porous beads called a "bed". The beads are coated with a substance that gives them an electrical charge. The buffer which passes through the bed has increasing concentrations of salts which cause the proteins to precipitate. The least strongly bound proteins are eluded first. In the polishing step, the protein is placed in "ultra pure H2O"; using gel filtration, proteins are separated by size. Other methods of separation are hydrophobic interaction chromotography and biological affinity chromotography.

History:

In 1975 the first transgenic animal was produced at Cold Spring Harbor by Jaenisch and Mintl. An ape transgene was implanted in a mouse and the protein was subsequently expressed but not passed on to offspring. The first transgenic strain of mice was produced by the same group in l977. In April of 1988, the "Harvard Oncomouse" was genetically engineered to contain a cancer-causing gene (an oncogene). This was the first mammal to be granted a patent; the project was sponsored by du Pont and in November of l988, sales of the patented mouse began. (These mice can be obtained from Charles River Biotechnology Services, Bausch and Lomb Co., Mass, at $50-100). Other transgenic mammals include: rabbits, goats, sheep, cattle, fish, poultry and pigs. Research on production of transgenic animals is being conducted in many other countries, primarily the U.K. Scientists in Russia have injected the gene for human growth hormone releasing factor into pig zygotes.

Application Of Transgenic Animals In General:

We can use these animals as models to study cancer and other human diseases. Mice models which have been developed include: cancer, Duchennes Muscular Dystrophy and Muscular Sclerosis.
We are able to increase our understanding of the human systems. Information gained is important to almost any aspect of modern biology: gene regulation, action of oncogenes, immune system, mammalian development.
We can engineer animals to be hypersensitive to mutagens and carcinogens for toxicology testing.
Human proteins and other pharmaceuticals can be produced in large quantities (as opposed to the small amounts produced by microbes) to use in research or treatment. This "molecular pharming" has given us experimental animals that could be very useful in treating human diseases. We have produced mice, pigs and cows that produce milk with proteins that dissolve blood clots. This tissue plasminogen activator (TPA) could be used to treat heart attack victims. A hydro-phobic membrane protein in milk of transgenic mice is a possible therapeutic for cystic fibrosis. Lactoferrin is an anti-bacterial protein pro-duced in milk of transgenic mammals that can be added to baby formula and used to treat immunosuppressed patients, such as AIDS babies.
Livestock can be engineered to have desirable traits such as disease resistance, faster growth, leaner meat.
It is also hoped that transgenic mammals will one day be used for xenografts. A xenograft is an organ of one species that is transplanted into a human. In October of l984, Dr. Bailey transplanted a baboon heart into "Baby Fae" who was born with a lethal malformation of her own heart. "Baby Fae" died 20 days after the operation; her body rejected the foreign tissue. It is no wonder that humans reject baboon tissue, since our lineages diverged 20-30 million years ago. Transplantation of chimpanzee organs into humans have proved to be more suc-cessful since our DNA is so similar. (Chimps and humans diverged 6-8 million years ago.) But chimps are an endangered species. A domesti-cated transgenic animal engineered with the proper proteins so that our immune systems would not recognize them as foreign could give us a sufficient supply of "spare parts".
Selective breeding of organisms to survive in distressed environments, such as transgenic fish to combat acid rain.

TECHNICAL PROBLEMS:

How to isolate and characterize chromosomal regulatory elements.
How to include these regulatory elements into the transgene.
How to produce markers so that early embryonic cells containing genes of interest can be identified and selected.
How to produce small animal models of diseases caused by complex, multiple gene changes. We cannot as yet "dial a gene" ; our present technique is still rather hit or miss.
How to control where in the animal's genome the transgene goes. Genetic material flanking that insert effects the expression of the gene.
How to determine if protein is being expressed. This can be done readily in the case of blood-borne proteins through electrophoresis or other assays. Those proteins expressed in milk cannot be tested until the animals are old enough to produce milk; and if the founder animal is a male such as Herman the bull...!
It is difficult to predict level of expression of the gene. Not every animal is going to be the golden goose. For instance, out of 152 microinjected bovine eggs, 112 were live born, of these, 3 females expressed the protein in milk at the rate of about 8 grams per liter, a fourth female expressed the protein at 35 grams per liter of milk.
Will these drugs receive FDA approval; so far none has been submitted for approval.

Issues On The Political And Legal Arena:

Moratoria on research with genetic engineering and transgenic mammals in particular have been the subject of many congressional hearings. The arguments against a moratorium are many. It could lead to secrecy and lack of public disclosure; competitiveness would be undercut and pro-gress in disease treatment could be hobbled. Since the private sector is now playing a larger role in funding agricultural research incentives should be protected.
Patenting of life is NOT a new issue. Thomas Jefferson back in l790 advised us that "ingenuity should receive a liberal encouragement". Patents on biotechnological processes go back many years. Louis Pasteur received a patent on a cultured strain of yeast for fermenting beer over a century ago; food patents date from the l800s; therapeutic patents were issued in l895. The first patent for an organism which was a result of recombinant DNA technology was awarded to Chakrabarty in l980. He had engineered a bacterium capable of breaking down multiple components of crude oil. The patent was rejected at first, however, on the grounds that microorganisms are products of nature and therefore not patentable subject matter. Since l980, many transgenic organisms have received biotech status. In order to be patented, an invention must be novel, useful and nonobvious; human intervention must be a necessary component of their creation. Patents are issued for a 17 year term. Others may not profit and the inventor must share his or her knowledge with others. Patents are not an automatic right to commercialize.
Some of the federal agencies using transgenic animals are: NIH in bio- medical research, USDA in livestock and veterinary research, NSF in laboratory research, AID in international research centers. No single federal policy governs all cases of genetically altered animals; the only policy specifically addressing use of genetically altered animals is the NIH guidelines for Research Involving Recombinant DNA Molecules.
There is also the question about whether the federal government should be financing biotechnology research.
The question might arise: Can humans be patented? The 14th amend-ment of the U.S. Constitution prohibits the ownership of one person by another. What about patent laws in other countries?

Ethical Concerns (Devil's Advocate Positions Are In Parenthesis)

Animal welfare groups feel that this could lead to increased animal suffering. Are we devaluing animal life by reducing it to a commercial commodity such as electric toasters and hula hoops? Many of the transgenes cause disease and suffering. (But we already eat them, wear them and put leashes on them. Historically, genetic material has been exchanged by classical hybridization; this new method takes less time, and allows for specific genes to be transmitted.)
Farm groups are afraid that the family farm could be hurt. This will surely boost the cost of production as a result of payment of royalties, concentration in production of animals. (But is the family farm any more sacred than the Ma and Pa grocery store or..?)
Environmental activists are concerned that the laws of nature might be upset. Are we violating species integrity? If an animal is changed substantially enough are we depriving it of its intrinsic mouseness or pigness. (But if that is the case can we justify the continued production of mules? Also, there already exists much fluidity and variability within a species naturally. At presently, relatively small numbers of genes are being manipulated (fewer than 20 in an animal that may contain from 50,000 Ñ 100,000 genes. Identity as a species is unlikely to be dis-rupted by this amount of change. Most of the domestic animals that are now the subjects of transgenic research are the products of generations of human manipulation.)
Religious leaders feel that Divine Law is being violated. (But who interprets "Divine" Law? )
Other countries have not permitted the patenting of animals, so our development and patenting of transgenic animals could lead to adverse economic implications for the Third World. (But our capitalistic system requires us to maintain the competitive edge.)
Many ethicists are concerned about how this issue affects our view of humanity. What will keep a warped mind from trying these techniques in humans? With genes and even blood and organs from other mam-mals, will our human spirit ("karma") be impaired? (But, humanity will benefit far more from this. We need to consider risks and benefits. Also, it is unlikely that any genes originating in animals will ever be inserted into humans, as we become able to synthesize genes. In the final analysis our DNA sequences have been shown to differ little from those of our close mammalian relatives. Diabetics have been treated for decades with insulin from pigs and cows.)
If many of these pigs with human genes are produced in the future, will they be made available for human consumption rather than fertilizers? If so, will this constitute cannibalism? See advocate objections for #5.
Religions which prohibit pig meat in their diets are concerned about the use of porcine produced pharmaceuticals. (But in Judaism, at least, pig may be used for purposes other than eating; in life and death situations, Kosher rules are set aside.)
Concerned coaches are afraid that this might lead to more "blood doping" by professional and amateur athletes. (But the development of life-giving drugs can not be stopped because there is a chance for abuse; otherwise no drugs would be developed.)
Jeremy Rifkin of the Foundation on Economic Trends and other groups such as the New Creation Institute of Missoula, Montana are concerned about all of these objections.

STUDENT FACT SHEET TO ACCOMPANY DNX ETHICAL QUESTION

The military estimates that having such a fluid would have saved the lives of 10,000 American soldiers in the Vietnam War. It will also be very useful in treating angioplasts since the cell-free liquid can go beyond the balloon. Other uses will be in radiation therapy where it will make the tumors more sensitive to radiation. "Naked" hemoglobin which is isolated may last only hours or days in the blood stream; it will serve therefore as only a temporary blood supply.

A unit (about a pint) of blood sells for $50. Concentrated blood sells for 175-200; DNX hemoglobin fluid would sell for 250. It is estimated that our nations need for blood could be supplied by 4 million donor pigs. Sixty million swine per year are raised for food.

Human clinical studies on artificial blood are to begin in l993. The company is anticipating spending 5 more years of research and testing. By the time it is ready to be marketed (after phase 2 and 3 of testing) the corporation will have spent $100-200 million on development of this blood substitute. The company expects to run at a loss until l996. DNX current revenues (Jan. 1992) are about $6.5 million; for more information contact Paul J. Schmitt, C.E.O. (tel: 609-520-0300).

The founder animal for Human Hemoglobin is alive in a total containment farm in Athens, Ohio along with generations of offspring; they say her name is Wilhelmina. Based on a 50 % expression rate, one pig should be able to produce the equivalent of l00 units of blood substitute per year. But so far the maximum expression of the gene has been l5%.

The National Institute of Child Health and Human Development (of N.I.H) awarded DNX $1.8 million to establish the National Transgenic Development facility in Princeton, N.J. DNX will do microinjecting for nonprofit research labs in exchange for licensing rights. The facility hopes to produce 100 animal models of human disease annually. A hundred researchers a year will pay $750 to DNX to produce founders with their gene of interest. NIH will subsidize the rest of the cost which is $7,000-10,000 per gene.

DNX has recently hired several staff members for xenograft research. They hope to grow "quasi-human" organs in pigs for transplants. In Dec. l990, there were 1,785 heart patients and l7,933 kidney patients on the national waiting list for organ transplants.

Pigs have a four month gestation. They are sexually mature at 6 months; their generation time is therefore about a year. 900 surgeries are performed each year. After surgeries at DNX, the sows are taken to the slaughter house. Since transgenic animals are not approved for consumption, all these pigs are "rendered" into fertilizer.

DNX, formerly Embryogen, injected extra growth hormone into pigs causing rapid growth, crippling arthritis, lethargy, low sex drive. This gene was not regulated so it was simply left on continuously. Experiments were stopped.

Scientists at Princeton University and Ohio University provided the expertise for the formation of this Corporation; they continue to sit on the scientific advisory board of the Corporation.

DNX-HIGH SCHOOL CONNECTION

Our high school student council has been awarded $1000 to invest in a promising new company. The council has looked into the financial promise of several companies and after careful review has recommended that we invest in DNX. A bioethics committee has been appointed to determine if this is an appropriate investment for our school. You will represent one of several special interests groups which will present your views to the bioethics committee. Follow the guidelines suggested and be as true to your role as possi-ble. Also limit your considerations to bioethical ones.

Suggested roles:

DNX BOARD, INDIVIDUALS WITH SICKLE CELL ANEMIA, ANIMAL RIGHTS GROUP, NEW CREATION INSTITUTE, FARM TO MARKET LEAN PORK, MEDICAL COMMUNITY.

GUIDELINES FOR PERSONAL POSITION ON DNX INVESTMENT

Where do you stand personally on investing your $1000 in DNX? Show your position on a continuum.
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YES---------------------------------------------------------------NO
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List in two separate columns why you would and why you wouldn't invest in DNX. Limit your lists to ethical concerns. State the asso-ciated value by each concern.
Write a paragraph stating your decision and your reasons for it.
Write another paragraph about the greatest dilemma you encountered while making this personal decision.

TECHNICAL QUESTIONS-DNX

(To accompany Vicki Glaser's article from Gen. Engineering News)

Describe in detail the various technologies that were required to develop this pig.
Why is there a need for porcine produced hemoglobin?
Why were pigs used in this development?
What are some of the technical hurdles mentioned in this article?
What are some other technical hurdles which could arise that were not mentioned in this article?
Suggest some names for the DNX pig/product.

TECHNICAL QUESTION-DNX

(TEACHER'S COPY)

Describe in detail the various technologies that were required to develop this pig.
A) RECOMBINANT DNA TECHNIQUES PRODUCTION OF TWO ALPHA GLOBIN GENES, ONE BETA CHAIN AND A LOCUS CONTROL REGION. (LCR ENSURES THAT NO MATTER WHERE THE RDNA PUTS ITSELF IN THE CHROMOSOMES OF THE PIG, IT WILL BE EXPRESSED. THE MORE COPIES OF DNA INTEGRATED, THE MORE EXPRESSION. LCR KEEPS HEMOGLOBIN LEVEL CONSTANT . HUMAN DNA INSERTS RANDOMLY WITHIN THE HOST CHROMOSOMES. 10% OF ANIMALS BORN CARRY RECOMBINANT FRAGMENTS.)
B) IVF
C) MICROINJECTION OF GENETIC MATERIAL. HUNDREDS OF COPIES OF GENE CONSTRUCT WERE MICROINJECTED INTO EACH PIG EMBRYO.
D) CHROMATOGRAPHIC PURIFICATION. ION EXCHANGE CHROMATOGRAPHIC PROCESS. CAN PRODUCE IN THE SCALE OF 1500 LITER RANGE FOR PURIFYING METRIC TON QUANTITIES. MORE THAN 95% OF THE HUMAN HEMO-GLOBIN IS RECOVERABLE. ADDITIONAL PURIFICATION REMOVES REMNANTS OF RED BLOOD CELLS AND PASTEURIZATION DESTROYS PATHOGENS.
E) ARTIFICIAL INSEMINATION. BREEDING TO PRODUCE HOMOZYGOUS ANIMALS DOUBLES THE COPY NUMBER.
F) REARING OF DISEASE FREE PIGS.
Why Is There A Need For Porcine Produced Hemoglobin?
A) CAN BE PRODUCED LARGE SCALE. A GOOD-SIZED PIG CAN PRODUCE 20 UNITS OF BLOOD SUBSTITUTE/YEAR. 650,000 TRANSGENIC PIGS WOULD BE NEEDED TO PRODUCE THE EST. 12-14 MILLION UNITS OF BLOOD TRANSFUSED IN THE U.S. ANNUALLY.
B) NO ALLERGIC REACTIONS BECAUSE IT WILL BE HUMAN HEMOGLOBIN.
C) NO AIDS OR HEPATITIS VIRUSES.
D) NO BLOOD TYPING NEEDED BECAUSE NO BLOOD CELLS WILL BE INVOLVED.
E) THOUGHT TO BE COST EFFECTIVE. RBC: $175-200/UNIT.
F) HAS A LONGER SHELF LIFE THAN HUMAN BLOOD
G) NO NEED FOR REFRIGERATION.
H) WELL SUITED FOR USE IN TRAUMA SITUATIONS
I) CAN BE USED FOR MAINTAINING ORGANS AND TISSUES FOR TRANSPLANTATION.
J) HEMOGLOBIN FOR TRANSFUSIONS (ONE UNIT OF PACKED RED BLOOD CELLS CONTAINS 50 G OF HEMOGLOBIN).
K) DO NOT CONTAIN ENDOTOXINS (HEMOGLOBIN PRODUCED BY MICROBIAL HOSTS DO).
L) RED BLOOD CELLS ARE MORE EFFICIENT FOR MAKING HEMOGLOBIN THAN BACTERIA (90% OF THEIR PROTEIN IS HEMOGLOBIN).
M) OTHER EXPERIMENTAL BLOOD SUBSTITUTES HAVE FAILED. (PERFLUOROCHEMICALS, PURIFIED COMPONENTS OF DONATED BLOOD, RECOMBINANT HEMOGLOBIN, BOVINE HEMOGLOBIN.) CROSSLINKING OF ALPHA GLOBIN CHAINS WAS OFTEN A PROBLEM.
Why Were Pigs Used In This Development?
A) REPRODUCE IN LITTERS THEREFORE CAN HAVE LARGE SCALE BREEDING.
B) PORCINE HEMOGLOBIN FUNCTIONS LIKE HUMAN HEMOGLOBIN SO PIGS CAN SURVIVE WITH THIS NEW HEMOGLOBIN IN THEM.
C) LARGE SIZE SO CAN SERVE AS EFFICIENT HEMOGLOBIN FACTORIES.
What Are Some Of The Technical Hurdles Mentioned In This Article?
A) PURIFICATION (FREE FROM STROMAL MATERIAL AND OTHER PROCINE PROTEINS).
B) CROSSLINKAGE AND STABILIZATION
C) EFFECT OF CROSSLINKAGE METHOD ON MEDICAL REACTIONS.
D) WILL THERE BE NEPHROTOXIC EFFECTS AS KIDNEYS ATTEMPT TO CLEAR HEMOGLOBIN.
E) SAFETY TRIALS IN HUMANS
F) EFFICACY TRIALS IN HUMANS
What Are Some Other Technical Hurdles Which Could Arise That Were Not Mentioned In This Article?
Hemoglobin Is Unstable And Half Life Is Only One Day So It Will Not Be A Suitable Substitute For All Uses.
Hemoglobin Is A Complex Protein. It Is Made Of Four Peptide Chains (2 Alpha Globins And 2 Beta Globins). Each Globin Contains An Iron Group Called Heme Which Can Bind With One Atom Of Oxygen. Hemoglobin Has Special Properties: (1) It Can Carry And Release Oxygen, (2) It Saturates With Oxygen At Atmospheric Pressure, And (3) A Gram Of It Can Absorb As Much As 1.3 Cc. Of Oxygen. Pure Hemoglobin In The Past However Has Not Proven To Be An Effective Substitute For Several Reasons: (L) Cell-Free Hemoglobin Lacks 2,3- Diphosphoglycerate And Therefore Has A Much Higher Affinity For Oxygen; (2) Cell-Free Hemoglobin Dissociates Readily Into Two Alph-Beta Globin Pairs (Dimers) Which Continue To Break Down Into Two Molecules; These Are Filtered In The Kidney And Lead To Kidney Damage. A System Has Been Devised To Prevent Hemoglobin From Dissociating Into Dimers But This Cross-Linking Procedure Also Reduces The Affinity For Oxygen. Another Procedure Which Achieved Stability And Acceptable Oxygen Affinity Proved To Have Toxic effects When It Was Tested In Humans.
The Gene For The Alpha Globin Is On The Human 16th Chromosome And The Gene For The Beta Globin Is On The 1lth.
Suggest Some Names For The DNX Pig/Product.
Acute Resuscitative Fluid, DNX Sausage - Its Like Having A Friend For Breakfast; DNX Bacon - That Old Familiar Taste; DNX Ham - We Put A Little Bit Of Ourselves Into Each Hog; It Takes All Kinds Of Critters To Make DNX Fritters; Purified Oxygenating Resuscitating Krud; Hamo-globin, Hemoglobin Augmentation For Mankind. (Compliments Of The Fine Minds That Gathered In July Of l992.)

References:

Jaenisch, R. "Transgenic Animals"; Science; June , l988; 1468-1473.

Glanz, James; "The Pharmaceutical Industry's New Star"; Research and Development; June, l992.

Glaser, Vicki; "DNX Corporation Develops Transgenic Pigs Capable of Manufacturing Human Hemoglobin," Genetic Engineering News; July/August; l991.

National Institutes of Health, Symposium on Transgenic Techniques in Medicine and Agriculture, Butterworth and Heineman, Boston, l991.

Sagoff, Mark; "Zuckerman's Dilemma"; Hastings Center Report ; Sept.- Oct. l991.

Smith, L.D.; DNX Corporation Company Report; Hambrecht and Quist Incorporated; One Bush Street; San Francisco, Calif. l992.

U.S. Congress, Office of Technology Assessment, New Developments in Biotechnology: Patenting Life; Washington, D.C. l988. (For sale by Superintendent of Documents, U.S. Government Printing Office, Washington, D.C., 20402.)

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