BIO149

WRITTEN REPORT
(Cloning, Stem Cells, Hybrid Cells)

Group 3:
Delmo, Carl Apoldom
Odia, Joseph Bien
Sarmiento, Tracy Mara
Timbreza, Jennah Camille

Cloning
What is cloning?

Cloning describes a number of different processes that can be used to produce genetically
identical copies of a biological entity. The copied material, which has the same genetic makeup
as the original, is referred to as a clone.
How Cloning is Done?
Artificial Embryo Twinning

Only done in a Petri dish

In the laboratory, the egg is usually fertilized by the sperm directly in the Petri dish. The
cells of a very early embryo are split from one another manually, so they can continue to
divide and mature on their own. The embryos are then placed in a surrogate mother.

Embryos are genetically identical.

Somatic Cell Nuclear Transfer

Germ cells- egg and the sperm cells.

Somatic cells- all other cells

Germ cells have one pair, or 23, chromosomes. Somatic cells have two pairs, or 46,
chromosomes.

SCNT begins with a scientist isolating a somatic cell and removing its nucleus

Nucleus contains all of our genetic information Then transfers nucleus to an egg that has
had its own nucleus removed.

The egg now has 46 chromosomes in its nucleus, which is just what happens when a
sperm (with 23 chromosomes) fertilizes a normal egg (also with 23 chromosomes)

Scientists then chemically stimulate the egg to believe it has been fertilized and it
begins to develop into an embryo. The embryo is then implanted into a surrogate mother.

The resulting organism is an exact clone of that which donated the somatic cell nucleus.

The History of Cloning

1885- First ever demonstration of artificial embryo twinning

Sea urchin
Hans Adolf Edward Dreisch

1902- Artificial embryo twinning in a vertebrate

Salamander
Hans Spemann
Strand of baby hair

1952- First successful nuclear transfer

Robert Briggs and Thomas King

Briggs and King transferred the nucleus from an early tadpole embryo into an enucleated
frog egg. The resulting cell developed into a tadpole.

1958- Nuclear transfer from a differentiated cell

Frog
John Gourdon
Transplanted the nucleus of a tadpole intestinal cell into an enucleated frog egg.

1975- First mammalian embryo created by nuclear transfer

Rabbit
J. Derek Bromhall
Glass pipette as a tiny straw
Transferred the nucleus from a rabbit embryo cell into an enucleated rabbit egg cell
Morula, or advanced embryo

1984- First mammal created by nuclear transfer

Sheep
Steen Willadsen
Separate one cell from an 8-cell lamb embryo
Used a small electrical shock to fuse it to an enucleated egg cell.
Placed the lamb embryos into the womb of surrogate mother sheep.
The result was the birth of three live lambs.

1987- Nuclear transfer from embryonic cell

Cow
Neal First, Randel Prather, Williard Eyestone
Methods very similar to those used by Willadsen on sheep
Produced two cloned calves (Fusion and Copy)

1996- Nuclear transfer from laboratory cells

Sheep

Ian Wilmut and Keith Campbell

Donor nuclei came from a slightly different source: cultured sheep cells

Transferred the nuclei from cultured cells into enucleated sheep egg cells.

Megan and Morag

1996- Dolly: First mammal created by somatic cell nuclear transfer

Sheep (Dolly)
Ian Wilmuth and Keith Campbell
Transferring the nucleus from an adult sheep's udder cell into an enucleated egg.
To produce Dolly, scientists used an udder cell from a six-year-old Finn Dorset
white sheep. They had to find a way to 'reprogram' the udder cells - to keep them alive
but stop them growing which they achieved by altering the growth medium (the soup
in which the cells were kept alive). Then they injected the cell into an unfertilised egg cell
which had had its nucleus removed, and made the cells fuse by using electrical pulses.

The unfertilised egg cell came from a Scottish Blackface ewe. When the research team
had managed to fuse the nucleus from the adult white sheep cell with the egg cell from
the black-faced sheep, they needed to make sure that the resulting cell would develop into
an embryo. They cultured it for six or seven days to see if it divided and developed
normally, before implanting it into a surrogate mother, another Scottish Blackface ewe.
Dolly had a white face.
From 277 cell fusions, 29 early embryos developed and were implanted into 13 surrogate
mothers. But only one pregnancy went to full term, and the 6.6 kg Finn Dorset lamb
6LLS (alias Dolly) was born after 148 days.

1997- First primate created by embryonic cell nuclear transfer

Rhesus monkey

Li Meng, John Ely, Richard Stouffer, and Don Wolf

Fused early-stage embryonic cells with enucleated monkey egg cells using a small
electrical shock. The resulting embryos were then implanted into surrogate mothers.

Out of 29 cloned embryos, two monkeys were born (Neti, Ditto)

1997- Nuclear transfer from genetically engineered laboratory cells

Sheep

Angelika Schnieke, Keith Campbell, Ian Wilmut

Human Factor IX (factor nine) gene into the genome of sheep skin cells grown in a
laboratory dish

To create the transgenic sheep, the scientists performed nuclear transfer using donor DNA
from the cultured transgenic cells.

Polly, a sheep that produced Factor IX protein in her milk.

1998- 1999- More mammals cloned by somatic cell nuclear transfer

Mice, cows, goats

2001- Endangered animals cloned by somatic cell nuclear transfer

Gaur, Mouflon, Bucardo (2009)

2007- Primate embryonic stem cells created by somatic cell nuclear transfer

Rhesus monkey

Shoukhrat Mitalipov and colleagues

Researchers took a cell from an adult monkey and fused it with an enucleated egg
cell. The embryo was allowed to develop for a time, then its cells were grown in a
culture dish. These cells, because they can differentiate to form any cell type, are
called embryonic stem cells.

2013- Human embryonic stem cells created by somatic cell nuclear transfer

Shoukhrat Mitalipov and colleagues

Create a human embryo that could be used as a source of embryonic stem cells
The resulting stem cell lines were specific to the patient they came from, a baby with a
rare genetic disorder.
Took a skin cell from the patient and fused it with a donated egg cell. Key to the success
of the experiment was modifications to the culture liquid in which the procedure was
done and to the series of electrical pulses used to stimulate the egg to begin dividing.

On December 22, 2001, the first domestic pet ever to be cloned was born: a kitten named CC,
pictured here next to her genetic donor, Rainbow.

X- Inactivation

In every cell, one X chromosome undergoes the process known as X-inactivation VERY
early in the embryonic stage.
X-inactivation is random. This means that one X may be active in some cells and inactive
in others. Once inactivated, the effect is permanent.
The two X chromosomes may have different alleles (i.e. different versions of the same
gene) on them. Which means, whichever X is active, the allele on that X will be
expressed. The allele on the inactive X will not be expressed.
One more thing to keep in mind: daughter cells resulting from cell division (e.g. mitosis)
will have the same inactivation pattern as their parent cell.

X- Inactivation in Rainbow and CC

Calico cats have coat color genes on the X chromosome. The gene has two alleles: one
for black fur and one for orange fur. Rainbow had one black allele on one X chromosome
and one orange allele on the other X chromosome.
CC, being Rainbows clone, also had one black allele and one orange allele.
The color that each cell expresses is dependent on which X is active and which is
inactive.
Rainbows random X-inactivation pattern gave her the coloring she has. Some cells
express black fur, others express orange fur.
The cell nucleus Rainbow donated to CC had already gone through X-inactivation. That
cell had the X for black fur active and the cell for orange fur inactive.
All of CCs cells originally came from that one donated cell and therefore could express
the gene for black fur, but not the gene for orange fur, even though she had an allele for
both.
As a result, CC has no orange fur at all!
To make it really complicated, further independent genetic effects result in white fur in
some places instead of black or orange. While the appearance of white fur is similar in
Rainbow and CC, it is not identical.

Why might we WANT to clone?

For medical purposes

Cloning animal models of disease study human diseases (especially geneticdiseases) in

large animal populations
Cloning stem cells for research stem cells may be used to repair damaged or diseased
tissues and organs
Large populations of genetically engineered cloned animals may be able to produce drugs
or proteins for medicine process called pharming

Revive endangered or extinct species

Reproduce deceased pets

Now being offered by one biotech company in United States

Any reason to clone a human?

Help infertile couples have children?

Replace a deceased child?

What are the RISKS of cloning?

High failure rate success rate is only about 0.1-3%

The egg and nucleus may not be compatible

The renucleated egg may be unable to divide or develop properly
Embryo implantation or the pregnancy itself may fail

Problems later in development

Many clones are born with large organs which leads to blood flow and breathing
problems (a condition known as Large Offspring Syndrome, or LOS)

Telomeric differences

Telomeres protect chromosomes from damage and naturally shorten every time DNA is
copied. This shortening is a natural aging process
Cloned animals begin life with an older organisms shortened telomeres and therefore
may prematurely age.

Abnormal gene expression

The transferred nucleus in the cell is NOT a natural embryo and thus cannot entirely
behave as one.

Stem cells
Stem cells are undifferentiated biological cells that can differentiate into specialized cells and
can divide (through mitosis) to produce more stem cells. They are found in multicellular organisms.
In mammals, there are two broad types of stem cells: embryonic stem cells, which are isolated from the inner
cell mass of blastocysts, and adult stem cells, which are found in various tissues. In adultorganisms, stem cells
and progenitor cells act as a repair system for the body, replenishing adult tissues. In a developing embryo,

stem cells can differentiate into all the specialized cellsectoderm, endoderm and mesoderm (see induced
pluripotent stem cells)but also maintain the normal turnover of regenerative organs, such as blood, skin, or
intestinal tissues.
There are three known accessible sources of autologous adult stem cells in humans:
1. Bone marrow, which requires extraction by harvesting, that is, drilling into bone (typically
the femur or iliac crest).
2. Adipose tissue (lipid cells), which requires extraction by liposuction.
3. Blood, which requires extraction through apheresis, wherein blood is drawn from the donor (similar to
a blood donation), and passed through a machine that extracts the stem cells and returns other
portions of the blood to the donor.
Stem cells can also be taken from umbilical cord blood just after birth. Of all stem cell types, autologous
harvesting involves the least risk. By definition, autologous cells are obtained from one's own body, just as one
may bank his or her own blood for elective surgical procedures.
Adult stem cells are frequently used in medical therapies, for example in bone marrow transplantation. Stem
cells can now beartificially grown and transformed (differentiated) into specialized cell types with
characteristics consistent with cells of various tissues such as muscles or nerves. Embryonic cell
lines and autologous embryonic stem cells generated through Somatic-cell nuclear
transfer or dedifferentiation have also been proposed as promising candidates for future therapies.[1] Research
into stem cells grew out of findings by Ernest A. McCulloch and James E. Till at the University of Toronto in
the 1960s.

Properties
The classical definition of a stem cell requires that it possess two properties:

Self-renewal: the ability to go through numerous cycles of cell division while maintaining the
undifferentiated state.

Potency: the capacity to differentiate into specialized cell types. In the strictest sense, this requires
stem cells to be either totipotent or pluripotentto be able to give rise to any mature cell type,
although multipotent or unipotent progenitor cells are sometimes referred to as stem cells. Apart from this
it is said that stem cell function is regulated in a feed back mechanism.

Self-renewal
Two mechanisms exist to ensure that a stem cell population is maintained:
1. Obligatory asymmetric replication: a stem cell divides into one mother cell that is identical to the
original stem cell, and another daughter cell that is differentiated.
2. Stochastic differentiation: when one stem cell develops into two differentiated daughter cells, another
stem cell undergoes mitosis and produces two stem cells identical to the original.

Potency definition
Potency specifies the differentiation potential (the potential to differentiate into different cell types) of the stem
cell.

Totipotent (a.k.a. omnipotent) stem cells can differentiate into embryonic and extraembryonic cell
types. Such cells can construct a complete, viable organism.[4] These cells are produced from the fusion of
an egg and sperm cell. Cells produced by the first few divisions of the fertilized egg are also totipotent.

Pluripotent stem cells are the descendants of totipotent cells and can differentiate into nearly all
cells, i.e. cells derived from any of the three germ layers.

Multipotent stem cells can differentiate into a number of cell types, but only those of a closely related
family of cells.

Oligopotent stem cells can differentiate into only a few cell types, such as lymphoid or myeloid stem
cells.

Unipotent cells can produce only one cell type, their own, but have the property of self-renewal, which
distinguishes them from non-stem cells (e.g. progenitor cells, muscle stem cells).

Identification
In practice, stem cells are identified by whether they can regenerate tissue. For example, the defining test for
bone marrow or hematopoietic stem cells (HSCs) is the ability to transplant the cells and save an individual
without HSCs. This demonstrates that the cells can produce new blood cells over a long term. It should also be
possible to isolate stem cells from the transplanted individual, which can themselves be transplanted into
another individual without HSCs, demonstrating that the stem cell was able to self-renew.
Properties of stem cells can be illustrated in vitro, using methods such as clonogenic assays, in which single
cells are assessed for their ability to differentiate and self-renew. Stem cells can also be isolated by their

possession of a distinctive set of cell surface markers. However, in vitro culture conditions can alter the
behavior of cells, making it unclear whether the cells will behave in a similar manner in vivo. There is
considerable debate as to whether some proposed adult cell populations are truly stem cells.

Stem cell therapy is the use of stem cells to treat or prevent a disease or condition. Bone marrow transplant is a
crude form of stem cell therapy that has been used clinically for many years without controversy. No stem cell
therapies other than bone marrow transplant are widely used.[71][72]
Research is underway to develop various sources for stem cells, and to apply stem cell treatments
for neurodegenerative diseases and conditions, diabetes, heart disease, and other conditions.[73]
In more recent years, with the ability of scientists to isolate and culture embryonic stem cells, and with
scientists' growing ability to create stem cells using somatic cell nuclear transfer and techniques to
created induced pluripotent stem cells, controversy has crept in, both related to abortion politics and to human
cloning.

Disadvantages
Stem cell treatments may require immunosuppression because of a requirement for radiation before the
transplant to remove the patient's previous cells, or because the patient's immune system may target the stem
cells. One approach to avoid the second possibility is to use stem cells from the same patient who is being
treated.
Pluripotency in certain stem cells could also make it difficult to obtain a specific cell type. It is also difficult to
obtain the exact cell type needed, because not all cells in a population differentiate uniformly. Undifferentiated
cells can create tissues other than desired types.
Some stem cells form tumors after transplantation; pluripotency is linked to tumor formation especially in
embryonic stem cells, fetal proper stem cells, induced pluripotent stem cells. Fetal proper stem cells form
tumors despite multipotency.
Hepatotoxicity and drug-induced liver injury account for a substantial number of failures of new drugs in
development and market withdrawal, highlighting the need for screening assays such as stem cell-derived
hepatocyte-like cells, that are capable of detecting toxicity early in the drug development process.

Research patents
Some of the fundamental patents covering human embryonic stem cells are owned by the Wisconsin Alumni
Research Foundation (WARF) - they are patents 5,843,780, 6,200,806, and 7,029,913 invented by James A.
Thomson. WARF does not enforce these patents against academic scientists, but does enforce them against
companies.
In 2006, a request for the US Patent and Trademark Office (USPTO) to re-examine the three patents was filed
by the Public Patent Foundation on behalf of its client, the non-profit patent-watchdog group Consumer
Watchdog (formerly the Foundation for Taxpayer and Consumer Rights). In the re-examination process, which
involves several rounds of discussion between the USTPO and the parties, the USPTO initially agreed with
Consumer Watchdog and rejected all the claims in all three patents, however in response, WARF amended the
claims of all three patents to make them more narrow, and in 2008 the USPTO found the amended claims in all
three patents to be patentable. The decision on one of the patents (7,029,913) was appealable, while the
decisions on the other two were not. Consumer Watchdog appealed the granting of the '913 patent to the
USTPO's Board of Patent Appeals and Interferences (BPAI) which granted the appeal, and in 2010 the BPAI
decided that the amended claims of the '913 patent were not patentable. However, WARF was able to re-open
prosecution of the case and did so, amending the claims of the '913 patent again to make them more narrow,
and in January 2013 the amended claims were allowed.
In July 2013, Consumer Watchdog announced that it would appeal the decision to allow the claims of the '913
patent to the US Court of Appeals for the Federal Circuit (CAFC), the federal appeals court that hears patent
cases. At a hearing in December 2013, the CAFC raised the question of whether Consumer Watchdog
had legal standing to appeal; the case could not proceed until that issue was resolved.

Hybridoma (Hybrid Cells)

Definition

A hybrid cell used as the basis for the production of antibodies in large amounts for
diagnostic or therapeutic use.

Hybridomas are immortalized cells derived from the fusion of B lymphoblasts with a
myeloma fusion partner. Some hybridomas in the ATCC collection are somatic cell
hybrids. These cells are capable of producing immunoglobulins that are specific for viral,
bacterial or cellular targets.

Hybridoma technology

The production of monoclonal antibodies was invented by Csar Milstein and Georges J.
F. Khler in 1975. They shared the Nobel Prize of 1984 for Medicine and Physiology

with Niels Kaj Jerne, who made other contributions to immunology. The
term hybridoma was coined by Leonard Herzenberg during his sabbatical in Csar
Milstein's laboratory in 1976/1977

Hybridoma technology is a technology of forming hybrid cell lines (called hybridomas)

by fusing an antibody-producing B cell with a myeloma (B cell cancer) cell that is
selected for its ability to grow in tissue culture and for an absence of antibody chain
synthesis. The antibodies produced by the hybridoma are all of a single specificity and
are therefore monoclonal antibodies (in contrast to polyclonal antibodies).

Method

Laboratory animals (mammals, e.g. mice) are first exposed to the antigen that an antibody
is to be generated against. Usually this is done by a series of injections of the antigen in
question, over the course of several weeks. These injections are typically followed by the
use of in vivo electroporation, which significantly enhances the immune response.

Once splenocytes are isolated from the mammal's spleen, the B cells are fused with
immortalised myeloma cells through electrofusion.

Fused cells are incubated in HAT medium (hypoxanthine-aminopterinthymidine medium) for roughly 10 to 14 days.

The next stage is a rapid primary screening process, which identifies and selects only
those hybridomas that produce antibodies of appropriate specificity.

Applications in terms of

Monoclonal Antibodies

Monoclonal antibodies can

and T cells, which is
types of leukemia.

distinguish subsets of B cells

helpful in identifying different

specific monoclonal
define cell surface
other cell types. This led to
of markers.

antibodies have been used to

markers on white blood cells and
the Cluster of differentiation series

In diagnostic
can be classified based on
markers, which reflect tissue or cellular genesis.

histopathology, tissues and organs

their expression of certain defined