Cloning Dolly & Micromanipulation

Topics of Discussion
What is cloning? Methods of cloning Dolly in detail Dollys probability Todays legality The future of cloning Ethical final questions

What is cloning?
Reproductive cloning- The entire animal is produced from a single cell by asexual reproduction. This would allow for the creation of a human being who is genetically identical to another. Therapeutic cloning- Broader use of the term cloning. Does not create a new genetically identical individual. Research includes therapy for human mitochondria disease and others that could replace damaged or diseased tissues without the risk of rejecting anothers tissue. Could create new skin tissue for burn patients.

Other types of cloning

Multiple copies of genes or gene fragments, repeating nucleotide sequences Single cell organisms, like bacteria and fungi. This includes fermentation processes for production of bread, beer, and wine. Entire plant asexual replication Natural cloning occurs in sexual reproduction, when the embryo splits in two to produce twins.

Methods of cloning
Embryo splitting- Artificially splitting a single embryo at a very early stage of development. In the natural process this would create twins. However, 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 cloning
Nuclear replacement- Genetic material (nucleus from embryonic, fetal, or adult cell) is removed and placed into an unfertilized egg or embryo, whose nucleus has been removed. In this case the nuclear genes remain the same but the mitochondria DNA would be different. This has the potential to create the clone of an adult organism as well as many clones at once.

What was Dolly?

In 1997 Dolly the sheep became the first vertebrate cloned from the cell of an adult animal. Not only was this a remarkable scientific breakthrough but it immediately gained interest and concern from around the world on the future of cloning technology as it would effect humans.

Dolly: The Cloning of a Sheep

Dolly the sheep was successfully cloned in Britain in 1996 by the scientist Ian Wilmut and was put down in February 2003 after developing a lung infection and arthritis. Dolly was a genetic copy of the Finn Dorset ewe. Her birth, more than 10 years ago showed that nuclei from specialized adult cells can be reprogrammed into all the cells of an organism. The technique that led to Dolly is called somatic cell nuclear transfer and has remained essentially unchanged over the last decade.

Dolly in detail
Dolly was cloned using the nuclear replacement method. Again the nucleus with chromosome sets is fused with an unfertilized egg whose nucleus has been removed. Motivating factor was that it could help to improve certain qualities in livestock. Dolly was not the first sheep to be created from nuclear replacement. Two genetically identical sheep, Megan and Morag were born in 1996 using the technique. The difference was that Dolly was derived from an adult sheep, and Megan and Morag were from a sheep embryo.

How to clone?

Dollys probability
Cells taken from a six-year-old Finnish Dorset ewe and cultured in a lab. 277 cells then fused with 277 unfertilized eggs (each with the nucleus removed) 29 viable reconstructed eggs survived and were implanted in surrogate Blackface ewes. 1 gave birth to Dolly 0.361% chance at onset, 3.4482% once implanted. In nature between 33-50% of fertilized eggs develop.

Cloning Dolly
Enucleate the eggs produced by Scottish Blackface ewes (female sheep).
Treat the ewes with gonadotropin-releasing hormone ( GnRH) to cause them to produce oocytes ready to

be fertilized. Like all mammals, these are arrested at metaphase of the second meiotic division (meiosis II). Plunge a micropipette into the egg over the polar body and suck out not only the polar body but the haploid pronucleus within the egg.

 Fuse each enucleated egg with a diploid cell growing in culture.

Cells from the mammary gland of an adult Finn Dorset ewe (they have white faces) are grown in tissue culture. Five days before use, the nutrient level in the culture is reduced so that the cells stop dividing and enter G0 of the cell cycle. Donor cells and enucleated recipient cells are placed together in culture. The cultures are exposed to pulses of electricity to
cause their respective plasma membranes to fuse; stimulate the resulting cell to begin mitosis (by mimicking the stimulus of fertilization).

 Culture the cells until they have grown into a morula (solid mass of cells) or even into a blastocyst (6 days). Transfer several of these into the uterus of each (of 13, in this case) Scottish Blackface ewes (previously treated with GnRH to prepare them for implantation. Wait (with your fingers crossed). The result: one ewe gave birth (148 days later) to Dolly.

Dolly with her foster mother

The Biotechnology of Reproductive Cloning

Even under the best of circumstances, the current technology of cloning is very inefficient.

Cloning provides the most direct demonstration that all cells of an individual share a common genetic blueprint.

How do we know that Dolly is not the progeny of an unsuspected mating of the foster mother?
She has a white face and the foster mother is a Scottish Blackface DNA fingerprinting reveals bands found in Finn Dorset sheep (the breed that supplied the mammary cells), not those of Scottish Blackface sheep

What about Dolly's telomeres?

It turns out that her telomeres are only 80% as long as those in a normal one-year-old sheep. The examination of DNA from Dolly's cells revealed that her telomeres were abnormally short. It is known that telomeres are sequences located at the end of each chromosome. These sequences protect DNA from degradation by exonucleases. In fact, telomeres are constantly degraded and restored.

 The balance becomes negative as the cells age, leading to a degradation of chromosomes and to cell death after about 50 multiplications. Dolly's telomeres were short but this was also the case for the donor cell line, which was derived from an old sheep and was cultured over a long period of time.

 It was prematurely suggested that cloning might generate old newborns. In all the cloned animals obtained after Dolly and in which DNA was examined, the length of telomeres was normal or longer than normal. This was also true for clones derived from a 17-yearold bull. It is also interesting to note that the two lambs born after Dolly was naturally fertilized have normal telomeres.

What about Dolly's mitochondria?

Although her nuclear genome came from the Finn Dorset ewe, her mitochondria came from cytoplasm of the Scottish Blackface ewe. Mitochondria carry their own genome and so with respect to the genes in mitochondrial DNA, she is not a clone of the Finn Dorset parent.

The Next Step?

Highly unlikely.

In Future Attempts at human cloning are viewed very unfavorably in the scientific community.

MICROMANIPULATION
The technique or practice of manipulating cells or tissues (as by microdissection or microinjection)

Micromanipulation
INTRODUCTION Since the birth of the first baby achieved through conception outside of the human body in 1978, the principles of "in vitro" (literally "in glass") fertilization and culture have remained the same - careful establishment and maintenance of a well-controlled, sterile environment in which the normal physiology of fertilization and early development can be played out relatively undisturbed to provide healthy embryos for transfer back into the body. During the ensuing two decades, much has been learned, however, about the tolerances of such a system and how this technique can be exploited to treat a widening range of infertility cases. There have been great strides made in development of more appropriate culture media that has enabled embryos to be grown for extended periods of time in culture. Surplus embryos and possibly eggs may now routinely be cryopreserved in liquid nitrogen for use in subsequent attempts at pregnancy. Fertilization itself is no longer a hit-and-miss affair with the advent of assisted fertilization through micromanipulation. Embryos can be micro-manipulated for cell biopsy to determine their genetic status as well as aid in their ability to implant through drilling into their outer shell (assisted hatching).

MICROMANIPULATION IN IVF THERAPY

Micromanipulation is the technique whereby sperm, eggs and embryos can be handled on an inverted microscope stage, performing minute procedures at the microscopic level via joysticks that hydraulically operate glass microtools.

MALE FACTOR INFERTILITY

Micromanipulation first saw clinical use in IVF for purposes of assisted fertilization in the treatment of male factor infertility, where fertilization potential was low in cases of poor sperm quality

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