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.
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
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.
Small groups: Begin ethical analysis (see Bill Smith's or Hastings Institute's model), record on melamine boards.
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".
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".)
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.
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.
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.
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.
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.
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.
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.
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.
DNX BOARD, INDIVIDUALS WITH SICKLE CELL ANEMIA, ANIMAL RIGHTS GROUP, NEW CREATION INSTITUTE, FARM TO MARKET LEAN PORK, MEDICAL COMMUNITY.
(To accompany Vicki Glaser's article from Gen. Engineering News)
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.)
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.
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.
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.
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
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.
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.)
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.)