Cell Revolution
Cell Revolution
Cell Revolution
Concept Outline
Photo Not Available
9e.1 Cloning animals, once thought impossible, isn’t.
The Challenge of Cloning. As recently as 1997, scientists
thought it was impossible to clone an adult animal. Then
researchers announced the successful cloning of a lamb from a
breast cell taken from an adult sheep.
Cloning Humans Is Not Going to Work Until a Key Problem
Is Solved. Cloning of animals usually fails for lack of proper
gene conditioning of the adult DNA used in the attempted
cloning.
The promise of gene engineering has had little impact on Although nuclear transplant experiments were attempted
farm animals. The difficulty in using genetic engineering to without success for decades, the technology continued to
improve livestock is in getting enough animals. Breeding advance. Finally in 1984, Steen Willadsen, a Danish embry-
genetically improved individuals produces offspring only ologist working in Texas, succeeded in cloning a sheep using
slowly, and recombination acts to undo the painstaking work a nucleus from a cell of an early embryo. This exciting result
of the genetic engineer. Ideally, one would like to “Xerox” was soon replicated by others in a host of other organisms,
many exact genetic copies of the desirable strain—but adult including cattle, pigs, and monkeys.
animals can’t be cloned. At least it was commonly accepted Only early embryo cells seemed to work, however.
that they couldn’t be. Now, in a surprising development, the Researchers became convinced that animal embryo cells
holy grail of agricultural genetic engineers seems within become irreversibly “committed” after the first few cell divi-
reach. In 1997, scientists announced the first successful sions. After that, they concluded, nuclei from differentiated
cloning of differentiated vertebrate tissue. animal cells could not be used to clone entire organisms.
We now know their conclusion to have been unwarrant-
ed. The key advance for unraveling this puzzle was made in
Spemann’s “Fantastical Experiment”
Scotland by geneticist Keith Campbell, a specialist in study-
The idea of cloning animals was first suggested in 1938 by ing the cell cycle of agricultural animals. By the early 1990s,
German embryologist Hans Spemann (called the “father of knowledge of how the cell cycle is controlled, advanced by
modern embryology”) who proposed what he called a “fan- cancer research, had led to an understanding that cells do not
tastical experiment”: remove the nucleus from an egg cell divide until conditions are appropriate. Just as a washing
and put in its place a nucleus from another cell. machine checks that the water has completely emptied
It was 14 years before technology advanced far enough for before initiating the spin cycle, so the cell checks that every-
anyone to take up Spemann’s challenge. In 1952, two thing needed is on hand before initiating cell division.
American scientists, Robert Briggs and T. J. King, used very Campbell reasoned, “Maybe the egg and the donated nucle-
fine pipettes to suck the nucleus from a frog egg (frog eggs us need to be at the same stage in the cell cycle.”
are unusually large, making the experiment feasible) and This proved to be a key insight. In 1994 researcher Neil
transfer a nucleus sucked from a body cell of an adult frog First, and in 1995 Campbell himself working with reproduc-
into its place. The experiment did not work when done this tive biologist Ian Wilmut, succeeded in cloning farm animals
way, but partial success was achieved 18 years later by the from advanced embryos by first starving the cells, so that
British developmental biologist John Gurdon. In 1970 he they paused at the beginning of the cell cycle at the G1
inserted nuclei from advanced toad embryos and adult small checkpoint. Two starved cells are thus synchronized at the
intestine into toad eggs. The toad eggs developed into tad- same point in the cell cycle.
poles, but almost all of them died before becoming adults.
FIGURE 9e.2
Wilmut’s animal cloning experiment. Wilmut combined a nucleus from a mammary cell and an egg cell (with its nucleus removed) to
successfully clone a sheep.
Early Success
That such gene transfer therapy can work was first demon-
strated in 1990. Two girls were cured of a rare blood disor-
der due to a defective gene for the enzyme adenosine deam- FIGURE 9e.4
inase. Scientists isolated working copies of this gene and Adenovirus (200,000×). This virus that causes the common cold
introduced them into bone marrow cells taken from the girls. has been used to carry healthy genes in clinical trials of gene ther-
The gene-modified bone marrow cells were allowed to pro- apy. Its use as a vector is problematic, however, as it is usually
liferate then were injected back into the girls. The girls attacked and destroyed by the immune system of the host. In addi-
tion, it can cause severe immune reactions and can insert into the
recovered and stayed healthy. For the first time, a genetic
host’s DNA at random places to cause mutations.
disorder was cured by gene therapy.
Adeno-associated
virus (AAV)
FIGURE 9E.5
Using gene therapy to cure a retinal degenerative disease in dogs. Recently, researchers were able to use genes from healthy dogs to
restore vision in dogs blinded by an inherited retinal degenerative disease. This disease also occurs in human infants and is caused by a
defective gene that leads to early vision loss, degeneration of the retinas, and blindness. In the gene therapy experiments, genes from dogs
without the disease were inserted into 3-month-old dogs that were known to carry the defective gene and that had been blind since birth.
Six weeks after the treatment, the dogs’ eyes were producing the normal form of the gene’s protein product, and by three months, tests
showed that the dogs’ vision was restored.
More Promising Vectors keys, scientists were able to greatly elevate their red blood
Within a few years, researchers had a much more promising cell counts, curing the monkeys of anemia—and they stayed
vector. This new gene carrier is a tiny parvovirus called cured.
adeno-associated virus (AAV). It has only two genes and needs A similar experiment using AAV cured dogs of a heredi-
adenovirus to replicate. To create a vector for gene transfer, tary disorder leading to retinal degeneration and blindness.
researchers remove both of the AAV genes. The shell that These dogs had a defective gene that produced a mutant
remains is still quite infective and can carry human genes into form of a protein associated with the retina of the eye, and
patients. Importantly, AAV always enters the human DNA at were blind. Injection of AAV bearing the needed gene into
the same place, a harmless location; thus, it does not produce the fluid-filled compartment behind the retina restored their
cancer-causing mutations. In addition, AAV does not elicit a sight (figure 9e.5).
strong immune response—cells infected with AAV are not Human clinical trials are now underway again. In 2000,
eliminated by a patient's immune system. Finally, AAV never scientists performed the first gene therapy experiment for
elicits a dangerously strong immune response, so it is safe to muscular dystrophy, injecting genes into a 35-year old South
administer AAV to patients. Dakota man. He is an early traveller on what is likely to
become a well-travelled therapeutic highway. Trials are also
underway for cystic fibrosis, rheumatoid arthritis, hemophil-
Success with the AAV Vector ia, and a wide variety of cancers. The way seems open, the
In 1999, AAV successfully cured anemia in rhesus monkeys. possibility of progress tantalizingly close.
In monkeys, humans, and other mammals, red blood cell
production is stimulated by a protein called erythropoietin New virus vectors like AAV avoid the problems of earli-
(EPO). People with anemia (that is, low red blood cell er vectors and offer promise of gene transfer therapy
counts), like dialysis patients, get regular injections of EPO. cures.
Using AAV to carry a souped-up EPO gene into the mon-
FIGURE 9e.6
Using embryonic stem cells to restore Photo Not Available
damaged tissue. Embryonic stem cells can
develop into any body tissue. Methods for
growing the tissue and using it to repair
damaged tissue in adults, such as the brain
cells of multiple sclerosis patients, heart
muscle, and spinal nerves, are being
developed.
Nucleus of
skin cell
Early embryo
Diabetic The nucleus from a
patient skin cell of a diabetic
patient is removed.
The nucleus is
removed from a The skin cell nucleus
Cell cleavage occurs
human egg cell. is inserted into the
as the embryo begins
enucleated human
to develop in vitro.
Human Enucleated egg cell.
egg cell egg cell
FIGURE 9e.8
How human embryos might be used for therapeutic cloning. Therapeutic cloning differs from reproductive DNA cloning in that
after the initial similar stages, the embryo is destroyed and its embryonic stem cells are extracted, grown in culture, and added to a tissue of
the individual who provided the DNA. In reproductive cloning, by contrast, the embryo is preserved to be implanted and grown to term in
a surrogate mother. It is this latter procedure that was done in cloning Dolly the sheep. Human cells were first cloned in November 2001
in a failed attempt to obtain stem cells for therapeutic cloning procedures such as outlined in this figure.
Diabetic
patient
Stem cells Embryonic stem cells The stem cells are developed
are extracted and into healthy pancreatic islet
grown in culture. cells needed by the patient.
Reproductive Cloning
Blastocyst
The embryo
reaches the
blastocyst stage.
Blastocyst
1 FERTILIZATION (6 days)
Ovary
2 IMPLANTATION
(7 days)
Egg Ovulation
FIGURE 9e.10
Four widely-held views about when human life begins.
(1) At fertilization; (2) At implantation; (3) at quickening;
(4) when independent survival is possible.
Heartbeat
(4 weeks)
When Does Human Life Begin?
The story of when human life begins has a checkered past.
Centuries before people knew of sperm and eggs, Aristotle
argued that the fusion creating a new person did not exist
until “quickening,” the first noticeable movements in a
woman’s womb. He reckoned quickening occurred 40 days
into pregnancy (18–20 weeks is the actual time). The 40-day
rule was picked up by Jewish and Muslim religions. In 1591,
Pope Gregory XIV supported this view of delayed anima-
tion and ensoulment. The Catholic Church did not reach its
current conclusion that life begins at fertilization until 1896,
3 “QUICKENING”
when Pope Pius IX condemned abortion at any age after the
(movement)
moment of conception. Many Jewish theologians now argue (20 weeks)
that life begins seven days into pregnancy, with implantation
of the embryo. Gene transcription starts even later (well
after stem cells are harvested), and many scientists feel
human individuality cannot be said to begin until then,
when the embryo starts to actually use its genes. The United
States Supreme Court takes the position that human life
begins much later, when the fetus becomes capable of inde-
pendent life if separated from the mother—roughly the
third trimester (figure 9e.10).
4 INDEPENDENT SURVIVAL POSSIBLE
(third trimester)
Embryonic stem cell research is quite controversial, as it
involves many ethical issues, not the least of which is
when human life begins.