DNA Technology and Genomics
Background Information
Cloning is the process of producing individuals that are genetically identical.
Cloning occurs in nature, seen when organisms such as bacteria, insects, and plants reproduce asexually.
Even in organisms that reproduce sexually, cloning can occur. i.e identical twins.
First reproductive cloning in 1952 in Amphibia.
First mammal cloned in 1997 in Edinburgh -Dolly the sheep.
Sheep, cattle (1998), mice (1998), goats (1999) and pigs (2000) have all been cloned.
DNA Technology
Recombinant DNA Technology or DNA Cloning
DNA fragment is
inserted and replicated by a bacterial plasmid.
http://wpcontent.answers.com/wikipedia/en/thumb/0/01/R ecombinant_formation_of_plasmids.svg/280px-
Recombinant_formation_of_plasmids.svg.png
Three types of Cloning Technologies:
Reproductive Cloning
Generate an organism that has the same DNA as another animal.
Example: Dolly
http://img.timeinc.net/time/daily/2008 /0811/dolly_sheep_1120.jpg
• Reproductive Cloning/Cloning Entire
Organisms.
Therapeutic Cloning
Stem cells that can be used to study human development and to treat
disease are harvested from a blastocyst.
http://library.thinkquest.org/06aug/01611/graphic s/technicalities-cloning/stemcell.png
Reproductive Cloning
Technology used to generate an animal that
has the same nuclear DNA as another currently
or previously existing animal.
Methods of Reproductive Cloning of Organisms
1- Embryo splitting-
Artificially splitting a single embryo at a very early stage of development. In the natural process this would create twins.
Because this is done at an early stage and there are usually less than eight cells you can only make a few clones. Both the nuclear genes and
mitochondria genes would be identical.
Methods of Reproductive Cloning of Organisms
2- Somatic Cells Nuclear Transfer
Genetic material (nucleus from embryonic, fetal, or adult cell) is removed and placed into an unfertilized egg, whose nucleus has been removed.
This has the potential to create the clone of an
adult organism as well as many clones at once.
Reproductive Cloning of Animals
Nuclear transplantation
Involves replacing nuclei of egg cells with nuclei from differentiated cells.
Has been used to clone a variety of animals.
• Scottish researchers cloned the first mammal in 1997
– Dolly, the sheep, was the product of their work.
• The procedure that produced Dolly is called
reproductive cloning.
Reproductive Cloning
Dolly was
successfully
born in 1997
Nuclear Transplantation Can Be Used to Clone Animals
Cultured mammary cells are semistarved ,arresting the cell cycle and causing dedifferentiation.
Steps for Somatic Cell Nuclear Transfer (Reproductive Cloning)
• Replacing the nucleus of an egg cell or a zygote with the nucleus of an adult somatic cell
• After 5 days, a blastocyst has formed
• Can be used for cloning (need surrogate mother)
Steps of Somatic Cell Nuclear Transfer
Nuclear material removed from donor egg
DNA inserted into the enucleated egg
Reconstituted zygote formed, equivalent to a fertilised ovum.
Potential to divide into a blastocyte
If implanted, develops into child genetically identical to the nuclear donor - Reproductive cloning.
In reproductive cloning the clone would be the identical twin of the donor
Other organisms have since been produced using this technique.
Reproductive Cloning Has Valuable Applications
Cloned sheep, mice, cats, horses, cows, and pigs
Can be used to study gene differences
Medical uses: can clone pigs with organs that can be transplanted into humans
Many animals experience health problems
Human cloning: ethically unacceptable (currently)
Reproduce animals with special qualities
Drug-producing animals
Result of breeding program (e.g. high milk producing cow)
Mass production of the best model organisms for studying human disease
Repopulate endangered animals
Problem: surrogate “mom” may be a different species than clone--less likely to be successful
Additional Applications of
Reproductive Cloning
Results of Nuclear Transfer Experiments
There were 277 failures before this nuclear
transfer technique succeeded.
What are the Risks of Reproductive Cloning?
Expensive and inefficient (<10% success rate).
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.
Many clones often die mysteriously, have poor health.
Small percentage of genes perform abnormally
Reproductive Cloning of Animals
Therapeutic Cloning/ Embryo Cloning
Instead of cloning entire organisms, there is therapeutic cloning
Stem cells are induced to turn into specific
tissue cells
Stem Cells
A stem cell is defined as:
A cell that can proliferate indefinitely and differentiate into a wide variety of cell types
Adult stem cells are found in bone marrow, isolated and encouraged to proliferate
Nuclear reprogramming – obtain ES cells by directly dedifferentiating normal body cells in vitro
Therapeutic Cloning
Therapeutic Cloning and Stem Cells
Therapeutic cloning
Produces embryonic stem cells (ES cells).
• Embryonic stem cells
– Can give rise to specific types of differentiated cells.
Procedure
1. Eggs are removed from the ovary of females and the nucleus is removed using a needle that is less than 2/10,000th of an inch wide.
2. DNA, usually from skin cells, is extracted and inserted into the ovum.
3. An electrical shock is applied to stimulate cell division.
4. The pre-embryo is allowed to divide for 5-14 days, after which, stem cells are extracted resulting in the death of the blastocyst.
5. The stem cells are encouraged to grow into specific organs which are then transferred to patients. Scientists have been able to
differentiate stem cells into most of the 220 cell types in the human body.
5 Day Old Blastocyst
http://www.brown.edu/Courses/BI0 032/gentherp/earlyIE1.htm
Therapeutic cloning
Blastocyte (embryo) cultured to produce an embryonic stem cell line
Excludes most blastocyte cells, effectively destroying the embryo
Undifferentiated embryonic stem (ES) cells can then be made to differentiate into precursor
cells.
Embryonic Stem Cells & Adult Stem Cells
Pluripotent Multipotent
Embryonic Stem Cells
Adult Stem Cells
Adult stem cells
Generate replacements for nondividing differentiated cells.
Are unlike embryonic stem cells, because they are partway along the road to
differentiation.
Therapeutic Cloning – Adult stem cells
Many benefits of embryonic stem cells can be achieved using adult stem cells.
Adult stem cells are demonstrating greater multipotency than expected
Adult stem cells hard to isolate and have restricted proliferation potential.
Range of cells they can be differentiated into is limited
Risks of using blood stem cells from a cancer patient’s own bone marrow to restore their immune system – some might be cancerous
Umbilical Cord Blood Banking
Umbilical cord blood and the placenta
Provide another source of stem cells.
Therapeutic Cloning:
Nuclear Transplantation Can Be Used to Generate Stem Cells
Replacing the nucleus of an egg cell or a zygote with the nucleus of an adult somatic cell
After 5 days, a blastocyst has formed
Can harvest embryonic stem cells for research (this destroys the embryo)
Day 5
Therapeutic Cloning:
Produce whole organs from single cells
Organs would be a genetic match.
Produce healthy cells to replace damaged cells (Alzheimer’s, Parkinson’s, etc..)
Genetically modified pigs for human organ donation.
NOT THERE YET!
Therapeutic Cloning Applications
Ways in which cloning may be expected to benefit mankind
• Use of embryonic stem cells to treat degenerative and autoimmune conditions such as Alzheimer’s Disease, Parkinson’s Disease, diabetes, heart failure, arthritis etc.
and to treat burns victims and spinal cord injuries.
Infertility treatment
Plastic, reconstructive and cosmetic surgery
Leukaemia and other cancers
Transplants – Kidney and liver
Reproductive - Therapeutic Cloning Process
http://content.nejm.org/content/vol351/issue27/images/large/02f1.jpeg
Induced Pluripotent Stem (iPS) Cells Genetically engineering new stem cells
Skin cells iPS cells
Pros and Cons to iPS cell technology
Pros:
Cells would be genetically identical to patient or donor of skin cells (no immune rejection!)
Do not need to use an embryo
Cons:
Cells would still have genetic defects
One of the pluripotency genes is a cancer gene
Viruses might insert genes in places we don’t want them (causing mutations)
Help this cloning works
