Cloning Fact ***

From: Michael Ragland (ragland37_at_webtv.net)
Date: 08/14/04 

Date: Sat, 14 Aug 2004 05:44:49 +0000 (UTC)

"Dolly or any other animal created using nuclear transfer technology is
not truly an identical clone of the donor animal. Only the clone's
chromosomal or nuclear DNA is the same as the donor. Some of the clone's
genetic materials come from the mitochondria in the cytoplasm of the
enucleated egg. Mitochondria, which are organelles that serve as power
sources to the cell, contain their own short segments of DNA. Acquired
mutations in mitochondrial DNA are believed to play an important role in
the aging process."

P.S. Nevertheless the clone's chromosomal DNA is the same as the donor's
and these clones are presented by both scientists and the media as
"genetically identical" and subsequently useful for disease studies and
nature/nurture studies.

The possibility of human cloning, raised when Scottish scientists at
Roslin Institute created the much-celebrated sheep "Dolly" (Nature 385,
810-13, 1997), aroused worldwide interest and concern because of its
scientific and ethical implications. The feat, cited by Science magazine
as the breakthrough of 1997, also generated uncertainty over the meaning
of "cloning" --an umbrella term traditionally used by scientists to
describe different processes for duplicating biological material.

What is cloning? Are there different types of cloning?

When the media report on cloning in the news, they are usually talking
about only one type called reproductive cloning. There are different
types of cloning however, and cloning technologies can be used for other
purposes besides producing the genetic twin of another organism. A basic
understanding of the different types of cloning is key to taking an
informed stance on current public policy issues and making the best
possible personal decisions. The following three types of cloning
technologies will be discussed: (1) recombinant DNA technology or DNA
cloning, (2) reproductive cloning, and (3) therapeutic cloning.

Recombinant DNA Technology or DNA Cloning
The terms "recombinant DNA technology," "DNA cloning," "molecular
cloning,"or "gene cloning" all refer to the same process: the transfer
of a DNA fragment of interest from one organism to a self-replicating
genetic element such as a bacterial plasmid. The DNA of interest can
then be propagated in a foreign host cell. This technology has been
around since the 1970s, and it has become a common practice in molecular
biology labs today.
Scientists studying a particular gene often use bacterial plasmids to
generate multiple copies of the same gene. Plasmids are self-replicating
extra-chromosomal circular DNA molecules, distinct from the normal
bacterial genome (see image to the right). Plasmids and other types of
cloning vectors are used by Human Genome Project researchers to copy
genes and other pieces of chromosomes to generate enough identical
material for further study.

To "clone a gene," a DNA fragment containing the gene of interest is
isolated from chromosomal DNA using restriction enzymes and then united
with a plasmid that has been cut with the same restriction enzymes. When
the fragment of chromosomal DNA is joined with its cloning vector in the
lab, it is called a "recombinant DNA molecule." Following introduction
into suitable host cells, the recombinant DNA can then be reproduced
along with the host cell DNA. See a diagram depicting this process.

Plasmids can carry up to 20,000 bp of foreign DNA. Besides bacterial
plasmids, some other cloning vectors include viruses, bacteria
artificial chromosomes (BACs), and yeast artificial chromosomes (YACs).
Cosmids are artificially constructed cloning vectors that carry up to 45
kb of foreign DNA and can be packaged in lambda phage particles for
infection into E. coli cells. BACs utilize the naturally occurring
F-factor plasmid found in E. coli to carry 100 to 300 kb DNA inserts. A
YAC is a functional chromosome derived from yeast that can carry up to 1
MB of foreign DNA.

Bacteria are most often used as the host cells for recombinant DNA
molecules, but yeast and mammalian cells also are used.

Reproductive Cloning

Celebrity Sheep Has Died at Age 6
Dolly, the first mammal to be cloned from adult DNA, was put down by
lethal injection Feb. 14, 2003. Prior to her death, Dolly had been
suffering from lung cancer and crippling arthritis. Although most Finn
Dorset sheep live to be 11 to 12 years of age, postmortem examination of
Dolly seemed to indicate that, other than her cancer and arthritis, she
appeared to be quite normal. The unnamed sheep from which Dolly was
cloned had died several years prior to her creation. Dolly was a mother
to six lambs, bred the old-fashioned way.

Image credit: Roslin Institute Image Library,
http://www.roslin.ac.uk/imagelibrary/

Reproductive cloning is a technology used to generate an animal that has
the same nuclear DNA as another currently or previously existing animal.
Dolly was created by reproductive cloning technology. In a process
called "somatic cell nuclear transfer" (SCNT), scientists transfer
genetic material from the nucleus of a donor adult cell to an egg whose
nucleus, and thus its genetic material, has been removed. The
reconstructed egg containing the DNA from a donor cell must be treated
with chemicals or electric current in order to stimulate cell division.
Once the cloned embryo reaches a suitable stage, it is transferred to
the uterus of a female host where it continues to develop until birth.

Dolly or any other animal created using nuclear transfer technology is
not truly an identical clone of the donor animal. Only the clone's
chromosomal or nuclear DNA is the same as the donor. Some of the clone's
genetic materials come from the mitochondria in the cytoplasm of the
enucleated egg. Mitochondria, which are organelles that serve as power
sources to the cell, contain their own short segments of DNA. Acquired
mutations in mitochondrial DNA are believed to play an important role in
the aging process.

Dolly's success is truly remarkable because it proved that the genetic
material from a specialized adult cell, such as an udder cell programmed
to express only those genes needed by udder cells, could be reprogrammed
to generate an entire new organism. Before this demonstration,
scientists believed that once a cell became specialized as a liver,
heart, udder, bone, or any other type of cell, the change was permanent
and other unneeded genes in the cell would become inactive. Some
scientists believe that errors or incompleteness in the reprogramming
process cause the high rates of death, deformity, and disability
observed among animal clones.

Therapeutic Cloning

Therapeutic cloning, also called "embryo cloning," is the production of
human embryos for use in research. The goal of this process is not to
create cloned human beings, but rather to harvest stem cells that can be
used to study human development and to treat disease. Stem cells are
important to biomedical researchers because they can be used to generate
virtually any type of specialized cell in the human body. Stem cells are
extracted from the egg after it has divided for 5 days. The egg at this
stage of development is called a blastocyst. The extraction process
destroys the embryo, which raises a variety of ethical concerns. Many
researchers hope that one day stem cells can be used to serve as
replacement cells to treat heart disease, Alzheimer's, cancer, and other
diseases. See more on the potential use of cloning in organ transplants.

In November 2001, scientists from Advanced Cell Technologies (ACT), a
biotechnology company in Massachusetts, announced that they had cloned
the first human embryos for the purpose of advancing therapeutic
research. To do this, they collected eggs from women's ovaries and then
removed the genetic material from these eggs with a needle less than
2/10,000th of an inch wide. A skin cell was inserted inside the
enucleated egg to serve as a new nucleus. The egg began to divide after
it was stimulated with a chemical called ionomycin. The results were
limited in success. Although this process was carried out with eight
eggs, only three began dividing, and only one was able to divide into
six cells before stopping.
How can cloning technologies be used?

Recombinant DNA technology is important for learning about other related
technologies, such as gene therapy, genetic engineering of organisms,
and sequencing genomes. Gene therapy can be used to treat certain
genetic conditions by introducing virus vectors that carry corrected
copies of faulty genes into the cells of a host organism. Genes from
different organisms that improve taste and nutritional value or provide
resistance to particular types of disease can be used to genetically
engineer food crops. See Genetically Modified Foods and Organisms for
more information. With genome sequencing, fragments of chromosomal DNA
must be inserted into different cloning vectors to generate fragments of
an appropriate size for sequencing. See a diagram on constructing clones
for sequencing.
If the low success rates can be improved (Dolly was only one success out
of 276 tries), reproductive cloning can be used to develop efficient
ways to reliably reproduce animals with special qualities. For example,
drug-producing animals or animals that have been genetically altered to
serve as models for studying human disease could be mass-produced.
Reproductive cloning also could be used to repopulate endangered animals
or animals that are difficult to breed. In 2001, the first clone of an
endangered wild animal was born, a wild ox called a gaur. The young gaur
died from an infection about 48 hours after its birth. In 2001,
scientists in Italy reported the successful cloning of a healthy baby
mouflon, an endangered wild sheep. The cloned mouflon is living at a
wildlife center in Sardinia. Other endangered species that are potential
candidates for cloning include the African bongo antelope, the Sumatran
tiger, and the giant panda. Cloning extinct animals presents a much
greater challenge to scientists because the egg and the surrogate needed
to create the cloned embryo would be of a species different from the
clone.

Therapeutic cloning technology may some day be used in humans to produce
whole organs from single cells or to produce healthy cells that can
replace damaged cells in degenerative diseases such as Alzheimer's or
Parkinson's. Much work still needs to be done before therapeutic cloning
can become a realistic option for the treatment of disorders.

What animals have been cloned?

Scientists have been cloning animals for many years. In 1952, the first
animal, a tadpole, was cloned. Before the creation of Dolly, the first
mammal cloned from the cell of an adult animal, clones were created from
embryonic cells. Since Dolly, researchers have cloned a number of large
and small animals including sheep, goats, cows, mice, pigs, cats,
rabbits, and a gaur. See Cloned Animals below. All these clones were
created using nuclear transfer technology.

Hundreds of cloned animals exist today, but the number of different
species is limited. Attempts at cloning certain species such as monkeys,
chickens, horses, and dogs, have been unsuccessful. Some species may be
more resistant to somatic cell nuclear transfer than others. The process
of stripping the nucleus from an egg cell and replacing it with the
nucleus of a donor cell is a traumatic one, and improvements in cloning
technologies may be needed before many species can be cloned
successfully.

Can organs be cloned for use in transplants?

Scientists hope that one day therapeutic cloning can be used to generate
tissues and organs for transplants. To do this, DNA would be extracted
from the person in need of a transplant and inserted into an enucleated
egg. After the egg containing the patient's DNA starts to divide,
embryonic stem cells that can be transformed into any type of tissue
would be harvested. The stem cells would be used to generate an organ or
tissue that is a genetic match to the recipient. In theory, the cloned
organ could then be transplanted into the patient without the risk of
tissue rejection. If organs could be generated from cloned human
embryos, the need for organ donation could be significantly reduced.

Many challenges must be overcome before "cloned organ" transplants
become reality. More effective technologies for creating human embryos,
harvesting stem cells, and producing organs from stem cells would have
to be developed. In 2001, scientists with the biotechnology company
Advanced Cell Technology (ACT) reported that they had cloned the first
human embryos; however, the only embryo to survive the cloning process
stopped developing after dividing into six cells. In February 2002,
scientists with the same biotech company reported that they had
successfully transplanted kidney-like organs into cows. The team of
researchers created a cloned cow embryo by removing the DNA from an egg
cell and then injecting the DNA from the skin cell of the donor cow's
ear. Since little is known about manipulating embryonic stem cells from
cows, the scientists let the cloned embryos develop into fetuses. The
scientists then harvested fetal tissue from the clones and transplanted
it into the donor cow. In the three months of observation following the
transplant, no sign of immune rejection was observed in the transplant
recipient.

Another potential application of cloning to organ transplants is the
creation of genetically modified pigs from which organs suitable for
human transplants could be harvested . The transplant of organs and
tissues from animals to humans is called xenotransplantation.
Why pigs? Primates would be a closer match genetically to humans, but
they are more difficult to clone and have a much lower rate of
reproduction. Of the animal species that have been cloned successfully,
pig tissues and organs are more similar to those of humans. To create a
"knock-out" pig, scientists must inactivate the genes that cause the
human immune system to reject an implanted pig organ. The genes are
knocked out in individual cells, which are then used to create clones
from which organs can be harvested. In 2002, a British biotechnology
company reported that it was the first to produce "double knock-out"
pigs that have been genetically engineered to lack both copies of a gene
involved in transplant rejection. More research is needed to study the
transplantation of organs from "knock-out" pigs to other animals.

What are the risks of cloning?

Reproductive cloning is expensive and highly inefficient. More than 90%
of cloning attempts fail to produce viable offspring. More than 100
nuclear transfer procedures could be required to produce one viable
clone. In addition to low success rates, cloned animals tend to have
more compromised immune function and higher rates of infection, tumor
growth, and other disorders. Japanese studies have shown that cloned
mice live in poor health and die early. About a third of the cloned
calves born alive have died young, and many of them were abnormally
large. Many cloned animals have not lived long enough to generate good
data about how clones age. Appearing healthy at a young age
unfortunately is not a good indicator of long term survival. Clones have
been known to die mysteriously. For example, Australia's first cloned
sheep appeared healthy and energetic on the day she died, and the
results from her autopsy failed to determine a cause of death.

In 2002, researchers at the Whitehead Institute for Biomedical Research
in Cambridge, Massachusetts, reported that the genomes of cloned mice
are compromised. In analyzing more than 10,000 liver and placenta cells
of cloned mice, they discovered that about 4% of genes function
abnormally. The abnormalities do not arise from mutations in the genes
but from changes in the normal activation or expression of certain
genes.

Problems also may result from programming errors in the genetic material
from a donor cell. When an embryo is created from the union of a sperm
and an egg, the embryo receives copies of most genes from both parents.
A process called "imprinting" chemically marks the DNA from the mother
and father so that only one copy of a gene (either the maternal or
paternal gene) is turned on. Defects in the genetic imprint of DNA from
a single donor cell may lead to some of the developmental abnormalities
of cloned embryos.
For more details on the risks associated with cloning, see the Cloning
Problems links below.

Should humans be cloned?

Physicians from the American Medical Association and scientists with the
American Association for the Advancement of Science have issued formal
public statements advising against human reproductive cloning.
Currently, the U.S. Congress is considering the passage of legislation
that could ban human cloning. See the Policy and Legislation links
below.
Due to the inefficiency of animal cloning (only about 1 or 2 viable
offspring for every 100 experiments) and the lack of understanding about
reproductive cloning, many scientists and physicians strongly believe
that it would be unethical to attempt to clone humans. Not only do most
attempts to clone mammals fail, about 30% of clones born alive are
affected with "large offspring syndrome" and other debilitating
conditions. Several cloned animals have died prematurely from infections
and other complications. The same problems would be expected in human
cloning. In addition, scientists do not know how cloning could impact
mental development. While factors such as intellect and mood may not be
as important for a cow or a mouse, they are crucial for the development
of healthy humans. With so many unknowns concerning reproductive
cloning, the attempt to clone humans at this time is considered
potentially dangerous and ethically irresponsible. See the Cloning
Ethics links below for more information about the human cloning debate.

By BBC News Online Science Editor Dr David Whitehouse

Tetra is a rhesus monkey and the first primate to be "cloned" using a
method that splits the original cells in an embryo to make multiple
identical animals.

It is a technique that could produce genetically-identical animals for
use in studies of human diseases like diabetes and Parkinson's.

The method is used often in cattle. A sperm and an egg are combined and
the resulting embryo allowed to split into two cells, then four, then
eight.

At the eight-cell stage, the embryo itself can be
split to produce four genetically-identical two-cell embryos.

This is the first time the technique has proved successful in monkeys.

"This is just artificial twinning," said Professor Gerald Schatten, from
the Oregon Regional Primate Research Center in Beaverton, US.
Dolly technique

In the Oregon research, it was hoped that each of the separated embryos
would develop to the stage where they could be transferred to a
surrogate mother for a "normal" pregnancy.
Splitting the embryo

But not all the split embryos survived - only two were implanted in
surrogates.
And only one had a normal pregnancy - Tetra was born 157 days later.

Although a limited number of clones can be produced this way, it is very
different from the technique used to produce Dolly the sheep, the first
cloned mammal and the subsequent clones of cattle, mice and goats.

Producing Dolly involved the transfer of the genetic material of an
adult into an empty cell sack, something that could in principle produce
as many clones as required.

The embryo splitting technique has pros and cons.

Because the cloning takes place after the formation of an embryo, the
exact characteristics of the animal that will develop from the embryo
are uncertain and will be exact copies only of themselves.

Only if embryos are produced using the Dolly technique will the animals
be genetically identical to another single (adult) individual.
Human ailments

The motivation of the researchers, who report their work in the journal
Science, is to produce genetically-invariable subjects for the study of
human diseases.

At the moment, monkeys are used in the study of human ailments, but the
genetic variation of groups of monkeys can make the evaluation of their
responses to procedures and medicines very difficult.

Having a batch of genetically-identical monkeys would simplify things
and would make interpreting results much more straightforward.
"In order to move discoveries from the laboratory bench to a patient's
bedside, we need to have genetically-identical animals that would
provide the information needed before these new therapies are tested on
people," said Professor Schatten. "Our contribution is to help provide
the genetically-identical models in which lifesaving cures can be
perfected."

Stem cells

The method Professor Schatten's team used is not yet very efficient. The
researchers made 368 embryos by splitting 107 embryos into two or four
pieces. Tetra was the only success.
The team now has four other pregnant monkeys which, if their babies make
it, are due to start delivering in May. The researchers plan to call two
of the genetically-identical twins Romulus and Rhesus.

Professor Schatten also intends to freeze split embryos.

These could later be harvested for stem cells, the so-called master
cells that can develop into any kind of cell in the body, and which
scientists hope one day to use as tissue transplants to treat diseases
such as diabetes and Parkinson's.

"It's uncertain whether intelligence has any long term survival value."
Stephen Hawking

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