CLONING

Lecture Notes for Biotechnology

What is Cloning? To most people, the term “cloning”

means making a copy of an individual.

In biology, cloning can have different uses depending on what is being copied.

Cloning Molecules and Cells DNA makes a copy when it

replicates to make two molecules A cell divides by mitosis to make

two identical daughter cells.

Cloning Organisms Botanists and home gardeners have

been cloning plants for centuries – take a “cutting” or piece of a plant and put in soil and it will form a new plant.

Animals that reproduce by asexual reproduction, such as small female freshwater crustaceans, produce offspring which are genetic copies of the mother

Cloning Vertebrate Animals In the 1960’s, John Gurdon

experimented with a technique called nuclear transplantation

He destroyed the nuclei of unfertilized eggs of the African clawed toad with UV light and replaced them with nuclei taken from intestinal cells of tadpoles of the same species

Gurdon (cont) A small percentage of the eggs

with the transplanted nuclei developed past the cleavage stage into tadpoles and adults

Later experiments using the transplanted nuclei from adult toad skin, kidney, heart and lung cells produced the same results

Cloning Mammals In 1997 Ian Wilmut and colleagues in

Scotland announced the birth of “Dolly” the first cloned mammal.

In a procedure similar to Gurdon’s, called somatic cell nuclear transfer, the lab group removed the nuclei from cells in the mammary gland of a sheep and placed them in enucleated eggs of another sheep.

This link shows images of somatic cell nuclear transfer (http://www.advancedcell.com/scnt.htm)

Dolly (cont) The eggs were stimulated to begin

dividing by treating them with either chemicals or electricity

Some of the eggs starting cleaving and were placed in the uteruses of other sheep. Only one attempt of 277 was successful, producing Dolly.

Dolly (cont) Dolly developed lung cancer and

arthritis and was euthanized in 2003 at the age of 6 years.

Most sheep of Dolly’s breed live to 11 or 12 years.

Other Cloned Mammals Since Dolly, other mammal species

have been successfully cloned. These include cow, goat, cat, pig, mule

and gaur. In many of these attempts, such as the

one producing CC the kitten, the donor nucleus is not removed from its cell but the donor cell is fused with an enucleated egg.

CC the Kitten Click on the link below to see Copy

Cat (CC) the cloned kitten (Nature, 2002)

CC has a different coat coloration, so she is not identical to the nucleus donor

http://www.nature.com/nature/journal/v415/n6874/fig_tab/nature723_F1.html

CC the Kitten CC is result of the fusion of a donor

cumulus cell and a recipient egg She is the sole live birth of 188

nuclear transfer procedures. 82 produced blastocysts.

Cloning Human Cells Researchers distinguish between

therapeutic and reproductive cloning In therapeutic cloning, the cloned

egg is allowed to divide for a few days and then the cells are separated from each other and saved as stem cells for potential use to treat diseases

Cloning Human Cells In 2001 a Worcester, MA company

called, ACT, announced the first human cloning. Of eight cloned eggs, only one made it to the six-cell stage

Later, a ban was put on this type of research by the US government if it was supported by public funds

Cloning Human Cells Research continues in other

countries and privately-funded labs in the US

Recently scientists in South Korea announced they successfully cloned a human embryo to the blastocyst stage and then separated the cells to begin stem cell lines

Cloning Human Cells In reproductive cloning, the cloned

egg would be allowed to divide the the blastocyst stage and the the embryo would be implanted into a woman’s uterus to continue development to birth

Cloning Human Cells A few years ago, a group called the

Raelians, announced the birth of a cloned human baby

They offered no scientific evidence to support their claim and it was dismissed by the scientific community

Are Clones Identical Copies? As shown by CC the kitten, clones

may not be identical to the nuclear donor

The uterine environment has an influence on development

Another reason may be the source of the mitochondria for the cloned embryo

Are Clones Identical Copies? Mitochondria have their own circular

strand of DNA coding for genes involved with their structure and function.

Mitochondrial genetic mutations cause several genetic diseases

Mitochondria are usually only maternally derived, the sperm does not supply any to a zygote.

Are Clones Identical Copies? In strict nuclear transfer, the

mitochondria would be supplied by the recipient cell not the nuclear DNA donor.

In the cell fusion technique, the mitochondria would come from both cells.

Potential Uses of Cloning Technology

Gene therapy Genetic engineering of organisms Sequencing genomes Reproductive cloning of animals to

produce some with special qualities

Other Potential Uses Reproductive cloning of animals to

repopulate endangered species. This has already been done with the gaur, a wild ox, and a mouflon, a wild sheep.

Therapeutic cloning to produce whole organs for transplants in humans

Therapeutic cloning to produce healthy cells to replace diseased cells

Risks of Cloning The technique rarely works and is

very expensive Cloned animals, like Dolly, do not

live long and have a variety of aliments

A third of cloned calves born alive die young and many are abnormally large

Risks of Cloning In cloned mice, 4% of the genes

function abnormally Genetic imprinting of sperm-

derived vs egg-derived genes is not possible

References http://www.ornl.gov/sci/techresour

ces/Human_Genome/elsi/cloning.shtml

Prentice, D. A. 2003. Stem Cells and Cloning. Benjamin Cummings

Cell biology: A cat cloned by nuclear transplantation. 2002. Nature. 415:859