Introduction to

Human Cloning

TIP 2

Group C

CLFS620 Modern Molecular Genetics

Focus Questions

Answer these questions as you view the Power point.

1. What is cloning?

2. What are the 3 types of human cloning?

3. Compare and contrast therapeutic

cloning and reproductive cloning.

4. What are ESC? What are sources of ESC?

5. Give an example of the usage of each of

the 3 types of human cloning.

What is Cloning?

CLONING is the creation of a copy of a gene or an entire organism using DNA

from an existing individual, so that the copy has the same genetic makeup as the original biological entity.

What is Human Cloning? Human cloning is the

creation of a genetically identical copy of an existing, or previously existing, human.

Potential uses of cloning

• Replacing organs and other tissues

• Infertility aid

• Treatment of human diseases

• Creating donor sources

• Gene therapy

• Reversing the aging process

Different Types of Human Cloning

DNA Cloning Therapeutic Cloning

Reproductive Cloning

also called gene cloning or recombinant DNA

produces copies of genes or segments of genes

the transfer of DNA pieces from one organism to a self-replicating genetic element

also called embryo cloning

produces a cloned embryo to create embryonic stem cells

cloning for the use of studying human development and treating disease

the type of cloning that produces a genetically identical human from another human

DNA Cloning

What is Gene (DNA) Cloning? (recombinant DNA)

Recombinant DNA transfer of a DNA fragment ofinterest from one organism to aself-replicating genetic element,such as a bacterial plasmid.

The DNA is then propagated inthe foreign host cell.

Basic Steps of Gene Cloning1) Restriction enzymes are

used to cut a fragment of DNA containing the gene to be cloned.

2) Fragments of the foreign DNA are inserted into plasmid vectors cut open with the same restriction enzyme or one which produces a match end.

3) DNA ligase seals the two DNA strands together to produce a recombinant DNA molecule.

Basic Steps of Gene Cloning4) The vector transports the gene

into a host cell (usually a bacterium).

5) Within the host cell the vector multiplies, producing numerous identical copies not only of itself but also of the gene that it carries.

6) When the host cell divides, copies of the recombinant DNA molecule are passed to the progeny and further vector replication takes place.

7) After a large number of cell divisions, a colony or clone of identical host cells is produced. Each cell in the clone contains one or more copies of the recombinant DNA molecule. The gene is cloned.

Uses of DNA Cloning

1. Isolation of a particular gene, part of a gene or region of a genome

2. Production of a desired RNA or protein molecule in large quantities

3. Increased production efficiency for commercially made enzymes and drugs

4. Modification of existing organisms so that they express a trait not previously encodedin the genome (transformation)

Uses of DNA Cloning

Therapeutic Cloning

What is Therapeutic Cloning?

• Therapeutic cloning refers to the medical procedure by which stem cells are harvested and grown to mature into transplantable organs or tissues.

• Creates new identical copies of an organism’s cells using their own DNA

Therapeutic CloningBasic Principle

1. Performed by removing healthy adult cells from a patient

2. reprogramming the cell’s nuclei

3. collecting and growing embryonic stem cell clones from the resulting blastocyst

4. and inducing these embryonic stem cell clones to differentiate into the stem cell or mature cell types required for transplantation.

Therapeutic Cloning is also called

Nuclear Transplantation or

Somatic Cell Nuclear Transfer (SCNT)

SCNT Process

Step 1: Remove the nucleus from an unfertilized egg cell (A) while using a suction pipette (B) to hold the egg cell steady and a glass needle (C) to remove the cell’s nucleus.

Step 2: Gently push the glass needle through the tough shell that surrounds the egg cell. The glass needle is used to remove the nucleus from within the egg.

http://www.pbs.org/wgbh/nova/sciencenow/3209/04-clon-nf.html

SCNT Process Step 3: The egg cell’s nucleus (A) has been released outside of the egg. This nuclear material will no longer be needed.

What remains is an “enucleated” egg (B). The enucleated egg contains certain molecules and other important factors that will help to establish embryonic stem cells.

Step 4: Inject the nucleus (red arrow) from a donor celldonor cell into the enucleated egg cell by easing the tip of the glass needle deep into the enucleated egg cell and depositing the donor nucleus.

SCNT Process Step 5: After completing the nuclear transfer, the unfertilized egg cell is “activated” using a chemical or electrical treatment that stimulates cellular division.

The first division results in two cells (left image), the next makes four cells, and so on, producing an embryo.

Step 6: The proliferating cells form a structure called a blastocyst within days, which is roughly the same size as the egg cell.

Cells taken from the blastocyst are embryonic stem cells (ESC).

Embryonic Stem Cells (ESC)

• are not implanted into the uterus

• are used to study development

• may be mixed with chemicals tohelp the cells take on different properties

• ultimately may be able tointroduce these cells into an adult(therapeutic cloning)

Embryonic Stem Cells can be cultured in different laboratory environments to develop into a specific cell type.

Culturedembryonicstem cells

(developing an ESC line)

Different cultureconditions

Liver cells

Nerve Cells

Muscle Cells

Different types ofdifferentiated cells

Embryonic Stem Cells (ESC)

• Embryonic stem cells (ESC) are cells that have yet to differentiate

• Unspecialized cells• Under certain conditions, can be induced to become

cells with specific functions (heart muscle cell, lymphocyte)

• Sources of ESC1. Adult Stem cells 2. In-vitro fertilization3. Umbilical Cord Blood (Core Blood) Stem cells

Adult Stem Cells“Adult” Stem Cells• Somewhat differentiated cells• Can develop into certain tissues, but not necessarily all tissues in the body

For example: Blood stem cells can develop into RBCs, WBCs, but not muscle cells

• Can be extracted from adults Ex. from bone marrow sample

Does Not require the creation of an embryo

Adult Stem CellsYou can also find these same type of stem cells in the blood system:

Peripheral Blood Stem Cells (PBSCs)

Used to treat leukemia, other cancers and various blood disorders

Less invasive than collecting bone marrow, but are sparse!

The bone marrow is the spongy core found in the bones and is a source of adult stem cells.

These stem cells are the precursor cells responsible for the formation of the blood cells (red blood cells, platelets, and white blood cells).

What is in Vitro Fertilization (IVF)?

• IVF is the process of fertilization by manually combining an egg and sperm in a laboratory setting

• Similar process to in-vitro fertilization

(“test-tube baby”), except embryonic

stems cells are not implanted into

mother

In vitro fertilizationSome procedures involved with IVF manually inject the sperm into the egg, and others simply allow fertilization to occur by

adding the sperm to the egg in the lab setting.

Why use IVF as a source of stem cells?

• According to a survey conducted in 2003, there are approximately 400,000 unwanted pre-embryos in the United States. (Hoffman, D.I., et al. 2003. Cryopreserved embryos in the United States and their availability for research. Fertility and Sterility 79: 1063-1069.)

• These may no longer be needed for fertility purposes and remain frozen or could be destroyed.

• They may be used in therapeutic cloning (without being implanted into a uterus).

Multipotent stem cell rich blood found in the umbilical cord has proven useful in treating the same types of health problems as those treated using bone marrow stem cells.

Umbilical Cord Blood Stem Cells

In 2005, there were more than 1,400 cord blood transplantations in adults, according to NETCORD, an international network that coordinates umbilical cord blood banks.

Differences between embryonic stem cells and adult stem cells:

-ESC can differentiate into any cell type (totipotent/pluripotent), while adult SC have already “committed” to a particular fate (multipotent).

Some Challenges in Research:-Adult stem cells are often present in only minute quantities and can therefore be difficult to isolate and purify. -There is also evidence that they may not have the same capacity to multiply as embryonic stem cells do. -They do not have the development potential of an ESC.-Finally, adult stem cells may contain more DNA abnormalities—caused by sunlight, toxins, and errors in making more DNA copies during the course of a lifetime. -These potential weaknesses might limit the usefulness of adult stem cells.

Therapeutic Cloning: Potential Application

• Repair a damaged tissue or group of cells that can't heal itself. – This might be accomplished by transplanting ESCs

into the damaged area and directing them to grow new, healthy tissue.

– It may also be possible to coax stem cells already in the body to work overtime and produce new tissue.

• To learn more about the causes of disease/improved research capabilities

• Tissue/Organ transplants• Potential solutions for currently incurable

degenerative diseases, like cancer• Substitute case for testing new drugs

Reproductive Cloning

What is Reproductive Cloning?

Making new, genetically identical copies of an organism, by using its own DNA

Could possibly create an “identical” twin, born years later

Process of Reproductive Cloninghttp://cmgm.stanford.edu/biochem118/images/Stem%20Cell%20Slides/08%20Cloning%20Procedures.jpg

Reproductive Cloning

• DNA originally from a mature somatic cell is added into an empty oocyte (egg).

• Once the egg has developed into an early-stage embryo inside a test-tube, it is implanted into the womb of an adult female animal.

• A cloned animal does not always look identical to the original animal.

What Animals Have Been Cloned?

Mice Cows

Sheep Chickens

Cat Deer

Dog Horse

Mule Ox

Pig Rabbit

Rat Rhesus

MonkeyHumans???

Ex. Dolly the Sheep (1996)

Efficiency of Reproductive Cloning

• Reproductive Cloning is a very inefficient use of technology.

• There are low success rates in the practice of cloning.

• More that 90% of cloning attempts fail to produce viable offspring. The cloning of Dolly took 276 attempts.

• Also, more than 100 nuclear transfer procedures could be required to produce one viable clone.

• Some studies show that after 60 cycles of cell division, stem cells can mutate and lead to cancer.

Source: http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml

Risks of Human Cloning

• Cloning tends to produce more compromised immune function and higher rates of infection, tumor growth, and other disorders.

• About 30% of all clones born alive are affected with large offspring syndrome and other debilitating conditions.

• Clones show premature aging and have shorter life spans.

• Also, there is no known facts on how cloning could impact mental development.

Sources: http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml

Ethical Issues of Human Cloning

-- Could allow for “manufactured” children with desired traits and characteristics

-- Unrealistic expectations of the clone’ssimilarity to the cloned individual

-- Could allow for the cloning of a deceasedindividual

-- May violate values of individual freedom, identity, and autonomy

-- Requires destruction of human embryos in a test tube

Comparing Therapeutic and Reproductive Cloning

Therapeutic cloning• Made in the same way as

reproductive cloning except the embryo is not implanted in a uterus

• Donor embryos are killed when stem cells are harvested from the embryos

• The stem cells are used to grow different types of tissues

Reproductive cloning• Made in the same way as

reproductive cloning with the embryo being implanted into a uterus to nurture a living individual

• Donor embryos are killed during the process.

• Cloned individuals have shorter lifespans and possible immune system malfunctions.

Acceptable or Not to the Public?

DNA or Gene Cloning Acceptable

Therapeutic Cloning Acceptable with reservations: Embryos are destroyed by stem cell extraction.

Reproductive Cloning Currently not Acceptable to clone humans

Works CitedCloning. The National Human Genome Research Institute. [cited 28 Apr 2009]. Available

from: http://www.genome.gov/pfv.cfm?pageID=25020028

Cloning in Focus. Genetic Science Learning Center at the University ofUtah. [cited 26 Apr 2009]. Available from: http://learn.genetics.utah.edu/units/cloning/

Perspectives. 2003. Center for Genetics and Society. Human Genetics in the Public Interest. [cited 1 May 2009]. Available from: http://www.genetics-and-society.org/perspectives/science.html

Reproductive Cloning. Center for Genetics and Society. [cited 26 Apr 2009]. Available from: http://geneticsandsociety.org/article.php?id=282

Therapeutic Use of Cell Nuclear Replacement: Therapeutic Cloning.Medical Research Council. [cited 26 Apr 2009]. Available from: http://www.reproductivecloning.net/therapeutic_cloning.pdf

United States Department of Energy. Office of Science. “Cloning Fact Sheet.” May 2009. Human Genome Project Information. Available from: http://www.ornl.gov/sci/techresources/Human_Genome/elsi/cloning.shtml