Unit IV
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Origin of organism cloning
Types of nuclear transfer
Cloning procedure in sheep
Application of cloning in conservation
Difference between therapeutic and
reproductive cloning
• Highlight ethical issues related to cloning
• Prospects of human cloning
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Two vector expression system
Directed mutagenesis
Transposone mutagenesis
Gene targeting
Site specific recombination
EBT 501, Genetic Engineering
Origins of organismal cloning in developmental
biology research on frogs; nuclear transfer
procedures and the cloning of sheep (Dolly) &
other mammals; applications in conservation;
therapeutic vs. reproductive cloning; ethical
issues and the prospects for human cloning;
Two-vector expression system; two-gene
expression vector, Directed mutagenesis;
transposon mutagenesis, Gene targeting, Site
specific recombination
Nuclear transfer procedures and
the cloning of sheep (Dolly) &
other mammals
Fertilization vs. Cloning (somatic cell
nuclear transfer, SCNT)
• Origins of organism cloning in
developmental biology research on frogs
Life Cycle of the frog
• The origin of nuclear transfer started in 1952
when R Briggs and TJ King performed first
successful nuclear transfer experiments in
frog species Rana pipens (Leopard frog)
• This was done by transplanting nuclei from the
blastula of the frog Rana pipens to an
enucleated eggs and obtaining a number of
normal embryos.
• Nuclear transfer is an embryological technique,
and involves removal of the nucleus from an egg
and replacement with the nucleus of another
donor cell. This experiment paved the way for
what we know today as Organism cloning
• Later, Gurdon 1986 showed that in Xenopus laevis
(African clawed frog), nuclei from various types of cell in
the swimming tadpole can be transplanted to an egg that
has been UV-irradiated to destroy the peripheral
chromosomes, and similar results were obtained.
• The important principle established was that, while
animal cells become irreversibly committed to their fate as
development proceeds, the nuclei of most cells still
retain all the genetic information required for the
entire developmental programme and can, under
appropriate circumstances, be reprogrammed by the
cytoplasm of the egg to recapitulate development
• Earlier the developmental stage at which nuclei are
isolated, the greater their potential to be reprogrammed.
• This allows animals with specific and desirable traits to be
propagated and have applications in farming to
• In 1989 Smith & Wilmut demonstrated for the
first time the Nuclear transfer in mammals in
which donor nuclei were obtained from the
morula or blastocyst-stage embryo and
transferred to an enucleated egg or oocyte (from
which the nucleus had been removed with a
pipette)
• Later on this procedure has been performed
successfully for rabbits and other farm animals
such as sheep, pigs and cows, which are far
more amenable to the process than mice.
• The donor nucleus can also be introduced by
promoting fusion between enucleated egg and a
somatic cell by giving a brief electric pulse, as it
activates embryonic development by stimulating
the mobilization of calcium ions.
• In 1995, Campbell and Wilmut produced two live lambs,
Megan and Morag by nuclear transfer from cultured
embryonic cells.
• This demonstrated the principle that mammalian nuclear
transfer was possible using a cultured cell line
• In 1996 Campbell and Wilmut reported the birth of Dolly,
following nuclear transfer from an adult mammary
epithelial cell line in
• This was the first mammal to be produced by nuclear
transfer from a differentiated adult cell, and aroused much
debate among both scientists and the public concerning the
possibility of human cloning
• However, the success rate was very low: only one of 250
transfer experiments produced a viable lamb.
• Similar transfer experiments have since been carried out in
mice, cows, pigs and goats
• The production of a transgenic mammal by
nuclear transfer from a transfected cell line was
first achieved by Schnieke in 1997, who
introduced the gene for human factor IX into
fetal sheep fibroblasts and transferred the nuclei
to enucleated eggs.
• The resulting sheep, Polly, produces the
recombinant protein in her milk and can
therefore be used as a bioreactor
• In 2000, McCreath succeeded in producing a
transgenic sheep by nuclear transfer from a
somatic cell whose genome had been specifically
modified by gene targeting. A foreign gene was
introduced into the COL1A1 locus and was
expressed at high levels in the lamb.
1999 – Richard Seed, M.D. announces plans to
develop nuclear transplantation technique for
humans
estimates 200,000 clones per year for
reproduction
2005 – Ian Wilmut group granted license (U.K) for
therapeutic cloning of human embryos
Problems with nuclear transfer:
very low efficiency (1%)
high frequency of developmental
abnormalities
• Difference between therapeutic and
reproductive cloning
• ethical issues related to cloning
• Prospects of human cloning
Therapeutic cloning (research cloning) is when
stem cells are extracted to grow into a piece of
human tissue which is encouraged to grow into a
human organ for transplant.
What are its uses?
- It is used for medical purposes, such as creating organs
to transplant into a patient in need of that organ. If
replacement organs are available to the sick and dying
people, countless numbers of lives could be saved.
Therapeutic cloning is a fast and efficient way to ‘repair’
damaged organs.
- Therapeutic cloning can be used to make insulinsecreting cells to cure for diabetes; nerve cells to cure
stroke or Parkinson’s disease
How is it done?
DNA is extracted from a human’s cell. The DNA
is inserted into a woman’s ovum and allowed to
develop and produce stem cells. The stem cells
are removed from the pre-embryo and are
treated to grown inyo whatever organ is needed.
Thus, the new organ is transplanted into the
patient.
Stem cells
Possible Benifits
-Cloned organs using therapeutic cloning is better than
organs donated by another person:
-No rejection of organ because the organ’s DNA would
match the patient’s DNA
-Patients do not need to wait for an organ donor to
donate his/her organs
-Brand new organs can work more efficiently than
donated organs
-Saving more lives, which otherwise would be lost due to
the waiting for a transplant
Potential Problems
-There are still many deficiencies/ethical
disputes in therapeutic cloning:
-Since therapeutic cloning is still in its
early stages of development stem cells
sometimes become mutated, thus
rejected by the recipient’s body.
-The production of stem cells needs to
become more efficient. So far we need
100 eggs to produce one usable stem
cell line.
-Many pro-life supporters believe that
human comes into existence at
conception. When the stem cells are
extracted the embryo dies, therefore
pro-life supporters believe that this is
murder.
Cloning (Somatic Cell Nuclear Transfer, SCNT)
Stem Cells
• Non-specialized cells that have the capacity to
divide in culture and to differentiate into more
mature cells with specialized functions
• Stem cells differ from other cells in three main
ways.
• First, they are “unspecialized,” meaning they
do not perform specialized functions, such as
the way heart muscle cells help blood flow or red
blood cells carry oxygen through the
bloodstream.
• Second, under certain conditions, they can be
transformed into cells with specialized
functions.
• Third, these cells are capable of reproducing
themselves over an extended period of time
Pluripotent cells
• Cells that are capable of self-renewal, and
have broad potential to differentiate into
multiple adult cell types.
• Pluripotent stem cells may be derived from
somatic cell nuclear transfer or from surplus
products of in vitro fertilization treatments
when such products are donated under
appropriate informed consent procedures.
Stem Cell Types
1. Adult Stem Cells
2. Embryonic Stem Cells
– Derived through fertilization
– Derived through nuclear transfer
Adult Stem Cell
• Undifferentiated cell found in a
differentiated tissue in an adult organism
that can renew itself and may, with certain
limitations, differentiate to yield all of the
specialized cell types of the tissue from
which it originated
Adult Stem Cells
• Derived from adults
• Identified in some organs
– Blood
– Eye
– Brain
– Skeletal muscle
– GI track (liver, pancreas, lumen)
– Skin
• Significant limitations
Current Clinical Uses of Adult Stem Cells
• Cancers—Lymphomas, multiple myeloma, leukemias, breast cancer,
neuroblastoma, renal cell carcinoma, ovarian cancer
• Autoimmune diseases—multiple sclerosis, systemic lupus,
rheumatoid arthritis, scleroderma, scleromyxedema, Crohn’s disease
• Anemias (incl. sickle cell anemia)
• Immunodeficiencies—including human gene therapy
• Bone/cartilage deformities—children with osteogenesis imperfecta
• Corneal scarring-generation of new corneas to restore sight
• Stroke—neural cell implants in clinical trials
• Repairing cardiac tissue after heart attack—bone marrow or
muscle stem cells from patient
• Parkinson’s—retinal stem cells, patient’s own neural stem cells,
injected growth factors
• Growth of new blood vessels—e.g., preventing gangrene
• Gastrointestinal epithelia—regenerate damaged ulcerous tissue
• Skin—grafts grown from hair follicle stem cells, after plucking a few
hairs from patient
• Wound healing—bone marrow stem cells stimulated skin healing
• Spinal cord injury—clinical trials currently in Portugal, Italy, S. Korea
Proposition 71: The California
Stem Cell Research and Cures Act
• Acknowledges that stem cell research has the
potential to produce new therapies
• States that “the federal government is not
providing adequate funding…”;
• Prop 71 will “close the critical funding gap”
• Supports “stem cell research, emphasizing
pluripotent stem cell and progenitor cell research
and other vital medical technologies, for the
development of live saving regenerative medical
treatments and cures”