Cloning 101

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Cloning 101

• Bio Tech
• March 17, 2009

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Medical Applications

• Consult list of 10 examples from our notes!

Dr. Cesario cultures stem cells for a stroke
patient -- http//www.mcg.edu
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The World View

• In 2003, the International Society for Stem Cell
  Researchers (ISSCR) drew about 700 scientists
• In 2005, more than 2000

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The UN gets involved bans all cloning
therapeutic reproductive

• Against the ban
• Belgium
• Canada
• China
• Denmark
• France
• New Zealand
• Singapore
• South Korea
• 26 other countries

• For the ban
• Australia
• Costa Rica
• Germany
• Ireland
• Italy
• Poland
• Switzerland
• US
• 76 others

37 countries abstained
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New Developments

• Obama plans to
• Lift funding ban
• Allow researchers to work on new stem cell lines
  that were off limits to everyone but private
  researchers
• These lines have genetic disorders
• http//www.bloomberg.com/apps/news?pid20601103s
  idayLc_3N41VkQreferus

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A Little Debate

• Video on the sides (approx. 30 min.)

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Cloning Timeline

• The Clone Zone

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Some terms and pictures you may want to know
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Some of the bigger experiments

• Cloning is often done with plants
• 1892 Driesch and his Sea Urchins
• 1918 vertebrates Spemann and his salamanders

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The Problem of Genomic Equivalence and Cell
Differentiation

• Genomic Equivalence
• All somatic cells of an adult organism are
  mitotic descendents of the original fertilized
  egg
• All somatic cells are, therefore, genetically
  identical
• The Problem
• If all cells are genetically identical, why
  arent they phenotypically identical?
• Can nucleus from an adult somatic cell take the
  place of the nucleus in the single fertilized egg
  AND effectively drive development?

Genome sum total of all genes in a given
organism
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Totipotency of Nuclei

• If all somatic cells of a given organism are
  genomically equivalent, the nucleus of each cell
  should be totipotent
• Totipotent
• Capable of directing the entire development of
  the organism
• Unlimited capability
• Give rise to pluripotent cells
• How can this be tested?
• Somatic Nuclear Transfer (commonly called
  cloning)
• Enucleate an activated but unfertilized egg
• Transplant a somatic cell nucleus into enucleated
  activated egg
• See what happens
• Does development proceed normally?

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First Successes With Somatic Nuclear Transfer

• Activation Pricking the animal pole with a
  glass needle simulates fertilization by a sperm
  and leads to cytoplasmic rearrangements necessary
  for embryonic development
• Enucleation Maternal chromosomes removed by
  glass needle

Carried out by Briggs and King, 1952, using Rana
pipiens (frog) eggs and donor blastulae
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First Successes With Somatic Nuclear Transfer

• Isolate nucleus from blastula donor cell
• Microinject donor nucleus into activated,
  enucleated egg

Carried out by Briggs and King, 1952, using Rana
pipiens (frog) eggs and donor blastulae
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Success of Somatic Nuclear Transfer Declines With
Older Somatic Donor Cells

• Interpretation
• As cells become differentiated, their nuclei
  lose their totipotency

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Along Came Dolly!Nuclei of Differentiated Cells
DO NOT Lose Their Totipotency

• Ian Wilmut, 1997
• First cloned mammal
• First clone produced using somatic nucleus from
  adult organism
• Source of nucleus Mammary gland cell of
  6-year-old pregnant ewe

Dolly 1st cloned mammal
Bonnie her lamb
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The Technique
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Clones Not Always Phenotypically Identical
CC A clone
Rainbow The somatic cell donor
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Back to the Original Problem

• All cells of adult animal are genomically
  equivalent
• Somatic cell nuclei are totipotent when placed in
  context of activated, enucleated egg
• So..
• Why arent all adult cells phenotypically
  identical?
• If they have identical genes, why do they
  differentiate into morphologically and
  functionally distinct cell types?

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Differential Gene Expression

• Not all genes are expressed at any given time in
  any given cell
• A cell will express only a subset of its genes
• The unique combo of genes expressed will
  determine (in large part) the cells phenotype
• What do we mean by the phrase gene expression?

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Lets review the steps of cloning

• Isolate the donor cells
• Remove and discard the nucleus from the egg cell
• Transfer somatic nucleus into the enucleated egg
  cell
• Stimulate cell division
• Implant embryo into surrogate mother

Click n Clone
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Quit Cloning Around!

• See the video for yourself!

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Therapeutic vs. Reproductive

• Therapeutic
• Also known as
• somatic cell nuclear transfer
• cell nuclear replacement
• research cloning
• embryo cloning
• Involves taking an enucleated egg and replacing
  that nucleus with DNA from the cell of another
  organism.
• The result is a blastocyst
• Almost identical DNA to the original organism.

• Reproductive
• Same basic transfer of genetic material
• The difference is that this egg is then inserted
  into a womb and allowed to divide indefinitely,
  and be born as an animal genetically identical to
  the parent.

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Therapeutic Cloning
Heart Cells
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Example Motor Neuron Disease (MND)

• A - Normally, each motor neuron controls a group
  of muscle fibers.
• B - when a motor neuron dies, a neighboring motor
  neuron can "sprout" new nerve endings to control
  the muscle fibers "orphaned" by the dying neuron,
  but these new connections aren't as strong as the
  original ones.

MDA Publications -- Vol. 5, No. 2 April 2000
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Is it Cloning?

• Play the Game

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Links

• Blood Vessel Repair
• Stem Cell Overview
• Anti-Aging

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Did you know.

• Human cloning is outlawed because if Chuck Norris
  were cloned, then it would be possible for a
  Chuck Norris roundhouse kick to meet another
  chuck Norris roundhouse kick. Physicists theorize
  that this contact would end the universe.

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What are Stem Cells

• Capacity to divide indefinitely
• Division always gives rise to a daughter stem
  cell and a more specialized daughter cell
• Pluripotent stem cells
• Present in early embryo
• Can generate ectoderm, endoderm, mesoderm, germ
  cells
• Not committed
• Committed stem cells
• Not pluripotent
• Reproduce themselves, plus give rise to more
  specialized cells

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Stem Cells
Non-equivalent cell division gives rise to 2
types of daughter cells
(Uncommitted)

• Progenitor (or precursor) cells
• NOT stem cells do not reproduce themselves
• Daughter cells can differentiate along several
  different pathways, depending on signals from
  cellular environment

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Blood Cell Formation
Gradual Restriction in Potency
(uncommitted) (committed)
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Is it safe?

• Embryonic stem (ES) cells are cultivated in
  "feeder layers" consisting of a nutrient material
  derived from live animal cells.
• Animal derived serum has also been used.
• A theoretical risk of viruses and other harmful
  agents being transmitted from the animal cells to
  the stem cells, and thus on to patients who
  receive stem cell therapy.

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Advantages and Disadvantages

• Advantages of ES
• Can be obtained in large quantities
• Very successful in animals
• Disadvantages
• Obtaining them is ethically ambiguous
• Stem cells would have to be genetically identical
  to the patient
• Possible risk that if the transplant dosage is
  not exact transplanted ES can form tumors
• The feeder cells and medium on which ES cells are
  grown may transmit viruses
• Adult Stem Cell Advantages
• Obtaining them is not ethically ambiguous
• No problem with genetic identity
• Disadvantages
• Relatively rare
• Not easily grown outside the body
• Certain organs do not have adult stem cells

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Is it safe?

• "The drive to be the first to provide cell lines
  for therapy could compromise safety for
  recipients and could lead this technology into
  the realms of quackery. Stem cell therapy needs
  to be nurtured safely and methodically to provide
  real benefit to patients in the future."

--http//news.bbc.co.uk
Cholera Outbreak Russia The World's News 1910
November 19
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The Scientists Evan Snyder

• Professor and Director
• Stem Cell Regeneration Program
• The Burnham Institute
• La Jolla, CA
• Progress is not going to be hitting a home run.
  Even thought we all swing for the fence, you can
  score just as effectively with bunts, singles,
  and occasionally stealing a base. If youre
  lucky, you hit a double.

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The Scientists Roger Pedersen

• Professor and Director
• Program in Stem Cell Biology
• Cambridge Stem Cell Institute
• University of Cambridge, UK
• When the building process for stem cell research
  matures a bit more, what will emerge will be
  something like the genome project, where theres
  a multinational, coordinated effort. There will
  be more coherent objectives instead of the kind
  of cottage industry mode that were in
  presently.

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The Scientists Hans Keirstead

• Assistant Professor of
• Anatomy and Neurobiology
• Reeve-Irvine Research
• Center
• University of CA, Irvine
• They dont want to embark on a four-to-seven
  year PhD program and become specialists in
  something the government is going to ban.

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Integrity?

• Feb 2004 Hwang Woo-suk's team declare they have
  created 30 cloned human embryos and extracted
  stem cells
• May 2005 Team says it has made stem cell lines
  from skin cells of 11 people
• Nov 2005 Hwang apologizes for using eggs from his
  own researchers
• 15 Dec 2005 A colleague claims stem cell research
  was faked
• 23 Dec 2005 Academic panel finds results of May
  2005 research were fabricated
• 10 Jan 2006 Panel finds 2004 work was also faked

Disgraced cloning expert Hwang Woo-suk
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When Does Life Begin?

• Its up to YOU!

Morula?
Sperm? Egg?
Zygote?
1st Heartbeat?
Just Born?
Blastocyst?
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Chew on This

• Be informed!
• Be educated!
• Be proactive!

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