GENETIC MANIPULATION OF ANIMALS

1. Traditional Techniques

-Pronuclear microinjection-Through virus-Isolation and transfusion of primordial germ cells

(PGCs), the embryonic cells that give rise to gametes-

2. Intracytoplasmic sperm injection uses sperm as passive carriers of recombinant DNA

3. Nuclear Tranfer technology

Somatic Cell Cloning

Introduction• Somatic-cell nuclear transfer (SCNT), or Somatic Cell

Cloning, is a laboratory technique for creating a clone

embryo with a donor nucleus. It can be used in

embryonic stem cell research, or, potentially,

in regenerative medicine where it is sometimes referred

to as "therapeutic cloning". It can also be used as the

first step in the process of reproductive cloning.

• Because somatic cell cloning is a cloning technique, it is

best to outline principles underlying the process of

cloning. Cloning is derived from the Greek word “klon”

which means a twig which can replicate itself and grows

eventually into a tree.

 

• To clone is to reproduce asexually or to make a copy or a set of copies of an organism following the fusion or insertion of a diploid nucleus into an oocyte.

• A true clone is an individual which has all the components that make up the individual, including nuclear genetic material (genome) and other maternally derived factors that are derived from a single unique embryo as a result of sexual reproduction.

• In the laboratory, cloning in mammals involves replacing the genetic material of an egg with the genetic material of a somatic cell from an embryo or adult which will eventually develop into a full organism or being ; this is a biological clone; an organism genetically identical to another organism.

• A major advance was made in 1995, when two live lambs, Megan and Morag (Fig. 13.7), were produced by nuclear transfer from cultured embryonic cells (Campbell et al. 1996). This demonstrated the principle that mammalian nuclear transfer was possible using a cultured cell line.

History

Fig. 13.7 Megan and Morag, the first sheep produced by nuclear transfer from cultured cells. Reproduced by kind permission of the Roslin Institute, Edinburgh.

• The same group later reported the birth of Dolly (Fig. 13.8), following nuclear transfer from an adult mammary epithelial cell line (Wilmut et al. 1997). 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 (see Johnson 1998).

It was suggested that a critical factor in the success of the experiment was the quiescent state of the cells in culture, allowing synchronization between the donor and recipient cell cycles (reviewed by Wilmut et al. 2002). For the production of Dolly, this was achieved by lowering the level of serum in the culture medium, causing the cells to withdraw from the cell cycle due to lack of growth factors. However, the success rate was very low: only one of 250 transfer experiments produced a viable lamb, a phenomenon that has been blamed on a lack of fundamental understanding of the nuclear reprogramming events that occur following transplantation (Shi et al. 2003). Similar transfer experiments have since been carried out in mice, cows, pigs, goats, cats, dogs, rabbits, mules, and rats using variations on the transfer methodology developed by Wilmut and colleagues.

In the case of Dolly, and possible attempts at human cloning, the American Medical Association defines cloning as the production of genetically identical organisms via somatic cell nuclear transfer, although a broader definition is often used to include the production of tissue and organs from cell or tissue cultures using stem cells. Somatic cell nuclear transfer refers to the transfer of the nucleus from an existing organism into an enucleated oocyte.

Somatic cell cloning is not equivalent to fertilsation

- In fertilization, the sperm and egg both contain one set of chromosomes. When the sperm and egg join, the resulting zygote ends up with two sets - one from the father (sperm) and one from the mother (egg).

In SCNT, the egg cell's single set of chromosomes is removed. It is replaced by the nucleus from a somatic cell, which already contains two complete sets of chromosomes. Therefore, in the resulting embryo, both sets of chromosomes come from the somatic cell

SCNT Process

• In the lab, a scientist extracts and discards the

nucleus of the egg cell, which is the part of the cell

that contains the egg donor's genes.

• The scientist then inserts the somatic cell from the

genetic donor into the egg and "fuses" the two

with electricity. The resulting fused egg contains

the genetic donor's DNA.

• The scientist stimulates the fused egg, which

"activates" the egg and causes it to divide just as an

egg would if it had been fertilized by a sperm cell in

conventional reproduction.

• The activated egg is then placed in a culture

medium. As cellular division continues over the

course of several days, a blastocyst (early-stage

embryo) forms.

• After about a week, an embryo transfer specialist

transfers the blastocyst to a recipient female

(sometimes referred to as "surrogate mother")

where it continues to develop. After a full-term

pregnancy, the recipient gives birth to an animal

that is essentially the identical twin of the genetic

donor.

There are two major techniques used in

somatic cell cloning:

The Roslin Technique is a variation of somatic cell nuclear transfer that

was developed by researchers at the Roslin Institute. The researchers used this method to create Dolly. In

this process, somatic cells (with nuclei in tact) are allowed to grow and divide and are then deprived of nutrients to induce the cells into a

suspended or dormant stage. An egg cell that has had its nucleus removed is then placed in close proximity to a

somatic cell and both cells are shocked with an electrical pulse. The

cells fuse and the egg is allow to develop into an embryo. The embryo

is then implanted into a surrogate.

The Honolulu Technique was developed by Dr. Teruhiko Wakayama

at the University of Hawaii.

In this method, the nucleus from a somatic

cell is removed and injected into an egg that

has had its nucleus removed.

The egg is bathed in a chemical solution and

cultured. The developing embryo is then implanted

into a surrogate and allowed to develop

• In this process, somatic cells (with nuclei in tact) are allowed to grow and divide and are then deprived of nutrients to induce the cells into a suspended or dormant stage.

In the case of dolly the sheep, the donor cell was transferred from 10% fetal calf serum to 0.5% fetal calf serum for five days, causing it to become inactive or enter the G0 stage. This allowed for enucleation and subsequent implantation of the somatic cell nucleus into an enucleated oocyte of a different organism. An egg cell that has had its

nucleus removed is then

placed in close proximity to a

somatic cell and both cells are

shocked with an electrical

pulse. The cells fuse and the

egg is allowed to develop into

an embryo.

The embryo is then implanted into a surrogate. This step is thought to mimic the stimulation normally provided by sperm during sexual reproduction.

A case study: the Life of Dolly

Birth- 5th July 1996

First Wave of Concern: At one year old, tests revealed that Dolly's

telomeres were shorter than those expected for

sheep of that age.

Dolly’s Life: In an attempt to allow Dolly to have as

normal as life as possible it was decided that she should

be allowed to breed

6 lambs: Her first lamb, named Bonnie,

was born in April 1998. The next year Dolly produced twin

lambs Sally and Rosie, and she gave birth to triplets Lucy, Darcy

and Cotton in the year after that.

Arthritis: In the autumn of 2001, at the height of

the Foot and Mouth outbreak in the UK, Dolly was seen to be walking stiffly

Dolly’s Final Illness: In January 2000, one of the cloned sheep,

Cedric, died. The post mortem revealed

that Cedric had died of sheep pulmonary adenomatosis (SPA).

Death: 14th of February

2003

Applications of Somatic Cell Cloning

• A key reason behind the usefulness of cloning is that by producing near-identical genetic copies of an organism, results are faster and more predictable than in previous reproductive techniques like artificial insemination, which involve costly and potentially harmful procedures such as cryopreservation.

• Many of these procedures require the use of stem cells.

Agriculture:

• SCNT ensures the rapid production of genetically modified herds or elite individuals with desirable traits, eg. For milk containing extra nutrients or meat more consistent in taste and quality.

• It also allows genetic conservation of local breeds with unique tolerance for regional diseases or local climates. This approach was used to clone the last surviving Enderby Island cow from mural granulosa cells.

• SCNT also allows spread of disease resistance faster than traditional techniques. For instance, herds of clones lacking the prion protein gene will no longer be susceptible to bovine spongiform encephaliti, also known as the Mad Cow disease.

Conservation:

• Conservation has been highlighted recently as an area where the SCNT technique may be useful. It may preserve and propagate endangered species that reproduce poorly in zoos until their habitats can be restored and populations reintroduced to the wild. Attempts have been made with the Giant Panda for instance. The technique allows maintenance or increase of the overall genetic diversity of a species by introducing new genes from preserved specimens or animals in other wild and captive populations of the same species back into a diminishing gene pool.

SCNT may even recreate extinct species, if viable tissues/cells have been banked or are available.

An example is the mammoth, where an intact animal was discovered frozen in the Tundra recently; the closely related elephant can be used as both oocyte donor as well as surrogate mother.

Medical Therapeutics:

• The greatest potential of the SCNT technique is in medical therapeutics and this is therapeutic cloning. The source cell can be human or animal, though the patient’s own cells will be the most likely source for therapeutic cloning.

Therapeutic cloning can be categorized into:• a. Replacement tissues & organs;• b. Prevention of immunological tissue rejection, to allow

for more successful organ transplantation;• c. Enhancement of immunological surveillance, to prevent

cancers; and• d. Gene therapy, to correct genetic defects by introducing

the functional gene (probably through stem cell re-population)

 • Clinical Applications include the ability to prevent, treat

and overcome: Aging, Disease, Cancers, Myocardial infractions and Genetic disorders amongst many others.

Clinical Applications can be grouped into two categories:

• Fine-tuning of existing strategies, eg. production of and screening for new drugs; and new strategies, also known as “cell-based therapy”

• Use is in a rapidly growing field of medicine, known as regeneration medicine. “Pharming” the production of pharmaceuticals by extracting and purifying desired molecules from the milk of genetically modified livestock is an example of the production of new drugs and proteins

The Success of SCNT procedures

• Cloning ensures the presence of a trans-gene by introducing the DNA into the somatic cell lines in culture instead of by the more traditional and tedious process of altering individual genotypes. Cattle producing insulin in their milk are an example.

• The first example published was Polly, another lamb cloned by the Roslin Institute. She was derived from fetal skin cells, genetically modified to contain a human gene. This has resulted in a valuable sheep that secretes human factor IX in its milk. The blood-clotting protein is extracted, purified, and used to treat haemophilia B.

SCNT is being used to produce human proteins for therapy. Human proteins are in great demand for the treatment of a variety of diseases. Whereas some can be purified from blood, this is expensive and runs the risk of contamination by HIV or Hepatitis C. Proteins can be produced in human cell culture but costs are very high and output small. Much larger quantities can be produced in bacteria or yeast but the proteins produced can be difficult to purify and they lack the appropriate posttranslational modifications that are needed for efficacy in vivo.

-SCNT can be used in cancer treatment by cloning cells from cancerous tissue and introducing specific characteristics leading to early cell death (eg. Short telomeres). Reintroducing the altered cells could decrease the capacity for division and replication in the tumour.

- SCNT can also be useful in biomedical research. Large animals can be genetically modified to carry genetic defects mimicking human illnesses such as cystic fibrosis. The similarities in organ size and life span allow for improved monitoring of factors such as the long-term consequences of treatment.

SCNT can also be used in xenotransplantation ( Transfer of living cells, from one species to another) The chronic shortage of organs means that only a fraction of patients who could benefit actually receive transplants. Genetically modified pigs are being developed as an alternative source of organs by a number of companies, though so far the modifications have been limited to adding genes.

Nuclear transfer will allow genes to be deleted from pigs and much attention is being directed to eliminating the alpha -galactosyl transferase gene. These encode an enzyme that creates carbohydrate groups which are attached to pig tissues and which would be largely responsible for the immediate rejection of an organ from a normal pig by a human patient.

Limitations

• A limitation of the SCNT technique for human therapeutic cloning is the need for human oocytes. Hence, the “universal donor” oocyte, Animal oocytes have been postulated for use with human somatic cells to create hybrid embryos, but this approach has not been accepted by many.

• Another limitation is the need for feeder cells to maintain the stem cells in an undifferentiated state.

• In SCNT, not all of the donor cell's genetic information is transferred, as the donor cell's mitochondria that contain their own mitochondrial DNA are left behind. The resulting hybrid cells retain those mitochondrial structures which originally belonged to the egg. As a consequence, clones such as Dolly that are born from SCNT are not perfect copies of the donor of the nucleus

• Stresses placed on both the egg cell and the introduced nucleuses are enormous, leading to a high loss in resulting cells. For example, Dolly the sheep was born after 277 eggs were used for SCNT, which created 29 viable embryos.

• Only three of these embryos survived until birth, and only one survived to adulthood. As the procedure currently cannot be automated, but has to be performed manually under a microscope, SCNT is very resource intensive. The biochemistry involved in reprogramming the differentiated somatic cell nucleus and activating the recipient egg is also far from understood

Ethical ConcernsSCNT AS UNETHICAL

• many believe that the use of somatic cell nuclear transfer in reproductive cloning defies the natural way of conceiving children.

• Reproductive SCNT would devalue the genetic distinctiveness of each individual. It would deprive the child of a sense of mystery or right to ignorance about his or her origin

• Religious groups do also believe that the use of technology to create a human clone is ‘playing with God’ and therefore is religiously, and morally, unacceptable

SCNT AS ETHICAL

• Somatic cell nuclear transfer procedures have been posed as ethical in 2 situations:

• Infertile couples who cannot otherwise be treated

• Couples at risk of passing a serious genetic disease on to their children. If both the male and female partners are carriers of autosomal-recessive disease traits, one partner’s somatic cell could be used to conceive. If one partner has an autosomal- dominant disease trait, the unaffected partner could provide the somatic cell.