cloning dolly

8/3/2019 Cloning Dolly

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Cloning Dolly

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Micromanipulation


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Topics of Discussion What is cloning?

Methods of cloning

Dolly in detail

Dollys probability

Todays legality The future of cloning

Ethical final questions


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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 couldreplace damaged or diseased tissues without the risk

of rejecting anothers tissue. Could create new skin

tissue for burn patients.


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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.


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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 beidentical.


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Methods of cloning

Nuclear replacement- Genetic material (nucleus

from embryonic, fetal, or adult cell) is removed and

placed into an unfertilized egg or embryo, whosenucleus 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.


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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 remarkablescientific breakthrough but it immediately

gained interest and concern from around the

world on the future of cloning technologyas it would effect humans.


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Dolly the sheep was successfully cloned in Britainin1996 by the scientistIan 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: The Cloning of a Sheep


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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 certainqualities 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 differencewas that Dolly was derived from an adult sheep, and Megan

and Morag were from a sheep embryo.


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How to clone?


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Dollys probabilityCells 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 Dolly0.361% chance at onset, 3.4482% once implanted.

In nature between 33-50% of fertilized eggs

develop.


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Cloning Dolly

Enucleate the eggs produced by Scottish Blackfaceewes (female sheep).

Treat the ewes with gonadotropin-releasing hormone (

GnRH)to cause them to produce oocytes ready tobe fertilized. Like all mammals, these arearrested at metaphase of the second meioticdivision (meiosis II).

Plunge a micropipette into the egg over thepolar body and suck out not only the polar bodybut the haploid pronucleus within the egg.
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Fuse each enucleated egg with a diploid cell growing inculture. 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 inculture.

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).
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Culture the cells until they have grown into amorula (solid mass of cells) or even into a

blastocyst (6 days).

Transfer several of these into the uterus of each (of13, in this case) Scottish Blackface ewes(previously treated with GnRH to prepare them forimplantation.

Wait (with your fingers crossed). The result: one ewe gave birth (148 days later) to

Dolly.
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Dolly with her foster mother


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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 commongenetic blueprint.


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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 ScottishBlackface sheep
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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 cellsrevealed 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.


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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.


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It was prematurely suggested that cloningmight generate old newborns.

In all the cloned animals obtained afterDolly 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 twolambs born after Dolly was naturallyfertilized have normal telomeres.


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What about Dolly's mitochondria?

Although her nuclear genome came from

the Finn Dorset ewe, her mitochondria

came from cytoplasm of the ScottishBlackface ewe. Mitochondria carry their

own genome and so with respect to the

genes in mitochondrial DNA, she is not aclone of the Finn Dorset parent.
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The Next Step?

Highly unlikely.

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


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MICROMANIPULATION

The technique or practice of manipulating

cells or tissues (as by microdissection or

microinjection)


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Micromanipulation

INTRODUCTIONSince the birth of the first baby achieved through conception outside of thehuman body in 1978, the principles of "in vitro" (literally "in glass") fertilizationand culture have remained the same - careful establishment and maintenance ofa well-controlled, sterile environment in which the normal physiology of

fertilization and early development can be played out relatively undisturbed toprovide healthy embryos for transfer back into the body. During the ensuing twodecades, much has been learned, however, about the tolerances of such a systemand how this technique can be exploited to treat a widening range of infertilitycases. There have been great strides made in development of more appropriateculture media that has enabled embryos to be grown for extended periods oftime 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 assistedfertilization through micromanipulation. Embryos can be micro-manipulated forcell biopsy to determine their genetic status as well as aid in their ability toimplant through drilling into their outer shell (assisted hatching).


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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.


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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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Questions or Comments?


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THANK YOU

cloning dolly

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