"Pharm Animals"

Sheep- Nancy- produces hujman alpha-1-anti-trypsin (AAT) in her milk.

AAT- a serum glycoportein helps keep air sacs inflate

20,000 people with inherited emphysema lack this protein.

Transgenic sheep make kgs of AAT in one milking session

Transgenic sheep-

fertility drugs for multiple ovulation

artificially insemination

Fertilized ova are washed out of the ewe (black sheep)

Microinjection of human AAT atached to a steep promoter.

Manipulated fertilized ova are put into a surrogate white sheep

Chimeras mated to produce homozygote- Milk tested for active protein

152 surrogates implanted with human AAT gene gave birth to 112 lambs. 1 male + 4 females were transgeneic. Only Nancy had active AAT ( 35 g/liter)

Transgenic Goats

Ethel- has human gene for factor VIII clotting factors for treatment of hemophilia A- 1 herd in Scotland produces enough for the world's hemo. Patients

Transgenic Boars-

Boar semen can be obtained anytime (up to about 1 pint).

Pharming Products

Cows- lactoferrin- added to infant formula to bind iron and prevent bacterial infection

Goats- tPA- breaks up blood clots

Pigs- hemoglobin- acts as a blood substitute

Rabbit erythopoieting- treats anemia from dialysis

Rat- human growth hormone- treats pituitary dwarfism

Sheept- Aat- hereditary emphysema

Plants- Genetically Engineered Food- In the United States ~ 70% of corn & 50% of soy bean crop are GM since 1996

Why-

Genetically engineered traits in crop plants

Resistance to insects, insecticides and herbicides

Slowed ripening

Larger fruits

Improved sweetness

Faster or more uniform growth

Additional nutrients

Easier processing

Pharmaceutical production

Insecticide production

Ability to fix nitrogen, decreasing fertilizer requirements

a. naked DNA into a plant cell nucleus by injecting via microinjection.

b. Ti plasmid (Tumor Inducing) from bacterium Agrobacterium tumefaciens

protoplasts- plants that have their cell walls removed to facilitate genetic engineering

Works well with dicots (cucumbers, squashes, beans, beets, tomatoes, potatoes, soybeans, cassava, & sunflower)

Monocots die from Ti plasmid infection.

Bacillus thurinigiensis- btr enzyme destroys stomach linings of certain insect larvae. Bt is introduced into corn so they are producing their own insecticides.

c. Monocots- (rice, corn, wheat, barley, oat, millet) gene transfer is by electroporation, microinjection, particle bombardment

Cloning People- First Publication of a cloning attempt by Scientific American Dec. 2000 issue

1.Eggs are coaxed to mature in a culture dish. Each has a remnant egg cell called the polar body and cumulus cells from the ovary clinging to it.

2. While an egg is held still with a pipette, a needle is used to drill through the zona pellucida, removing a plug.

3. After ejecting the zona plug, the needle is inserted back in the egg through the hole to withdraw and discard the polar body and the egg's genetic material.

4. A cumulus cell from another egg is taken up into the needle. Cells called fibroblasts (or their nuclei) can also be used in this step.

5. The cumulus cell is injected deep into the egg that has been stripped of its genetic material.

6. The injected egg is exposed to a mixture of chemicals and growth factors designed to activate it to divide.

7. After roughly 24 hours, the activated egg begins dividing. The cells contain genetic material only from the injected cumulus cell.

8. By the fourth or fifth day, a hollow ball of roughly 100 cells has formed. It holds a clump of cells called the inner cell mass that contains stem cells.

9. The blastocyst is broken open, and the inner cell mass is grown in a culture dish to yield stem cells.

The stem cells, in turn, can be coaxed to grow into a variety of cells that might one day be injected into patients.

Total of 71 eggs from 7 volunteers (paid $4000- $40 hour)
Of 8 eggs we injected with cumulus cells, 2 divided to form early embryos of 4 cells–and one progressed to at least 6 cells–before growth stopped.

Why did they do it?

Therapeutic cloning–use the genetic material from patients’ own cells to cure diseases

Examples: generate pancreatic islets to treat diabetes or nerve cells to repair damaged spinal cords

Therapeutic cloning is distinct from reproductive cloning, which aims to implant a cloned embryo into a woman’s uterus leading to the birth of a cloned baby.

Reproductive cloning has potential risks to both mother and fetus Advance Cell Technology supports restriction of cloning for reproductive purposes until the safety and ethical issues surrounding it are resolved.

Is it ethical to clone humans?

If a cloned organism were implanted into a womb, as was done in the case of Dolly the sheep, it could possibly go on to full development and birth.

Because of this potential, some would argue that the organism produced in human therapeutic cloning experiments is the equivalent of any ordinary human embryo and merits the same degree of respect and protection.

Some people feel that cloning is ethically wrong-

incidence of deaths and birth defects in cloned animals;

improper gene expression and imprinting;

possible psychological risks to children produced in families in which a parent may also be a child’s genetic twin. Cloned children may face unrealistic expectations to live up to the achievements of their genetic predecessor;societies decide to replicate a limited number of desired genomes on a large scale for military or other purposes.

In opposition to this, some people hail the prospect of cloning

Who wants to pursue Reproductive Cloning?

Panos Zavos (University of Kenturky) and Severino Antinori (Rome-based fertility clinic).

Antinori has helped many post-menopausal women become pregnant, including at least one woman who gave birth in her 60s.

At a conference on human cloning at the National Academy of Sciences last August, Zavos said that he and Antinori would work to help couples in which the man did not produce viable sperm reproduce via cloning.

The two announced they would have pregnancies by the end of this year. Their claim is credible: both have extensive expertise in fertility and access to potentially interested couples.

Clonaid/The Raëlians. The Raëlians are a religious group that believes that humans descended from extraterrestrials

The Raëlian company, Clonaid, led by Chemist Brigitte Boisselier, told the National Academy meeting last August that Clonaid had hundreds of women willing to contribute eggs for use in cloning–an important first step toward success.

Boisselier argued that people should have the liberty to reproduce how they want, whether by combining their genetic material with another person's through sex or in vitro fertilization or by using only their own genetic material to create a clone.

Richard G. Seed. A physicist with an interest in embryology based near Chicago.

Seed is an advocate of cloning to treat severe infertility as well as to "replace a lost loved one with a twin."

He claims he will have three pregnancies before 2002. He is known to have attracted a skilled reproductive scientist from China to aid in his efforts, but he does not appear to have the other resources he would need to succeed.

Alternates to cloning- Parthenogenesis

Induce human eggs to divide into early embryos without being fertilized by a sperm or being enucleated and injected with a donor cell.
Normally mature eggs and sperm are haploid. If activated before Meiosis II, they still retain a full set of genes.
Stem cells would not be rejected because very similar (although not identical - WHY?)
Easier to perform w/ females, but tech. possible w/ males by fusing enucleated sperm

Advanced Cell Tehcnology exposed 22 eggs to chemicals that changed the concentration of charged atoms called ions inside the cells.

After 5 days of culture 6 eggs had developed into what appeared to be blastocysts. None clearly contained the so-called inner cell mass that yields stem cells.

Preimplantation Genetic Screening
Cells can be removed from early embryos and screened for genetic and chromosomal abnormalities. See figure 20.7

$10,000 fee (probably not covered by health insurance)

Legalities-Will therapeutic cloning end up being against the law?

On July 31, 2001- House of Representatives voted for a broad ban on human cloning
Prohibit reproductive & therapeutic cloning
Penalties - up to 10 years in prison & fines of $1 million for anyone who generates cloned human embryos.
An amendment that would have allowed therapeutic cloning failed.

The Weldon/Stupak bill (in Senate by early 2002) opposes human cloning for any purpose (endorsed by the Bush administration).

U.K. Parliament (2000) altered its Human Fertilization & Embryology Act of 1990 to specifically allow human therapeutic cloning. But last November anti-abortion activists succeeded in having the provision struck down on the grounds that cloning does not involve an embryo created by the union of an egg and a sperm and therefore cannot be included under the act.

Aug.2001 President George Bush barred the use of federal funds for research involving stem cells derived from embryos,including those generated using cloning. The bar permits federally funded scientists to experiment only with pre-existing stem cell cultures, or lines.