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Cloning 101

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Title: Cloning 101


1
Cloning 101
  • Bio Tech
  • March 17, 2009

2
Medical Applications
  • Consult list of 10 examples from our notes!

Dr. Cesario cultures stem cells for a stroke
patient -- http//www.mcg.edu
3
The World View
  • In 2003, the International Society for Stem Cell
    Researchers (ISSCR) drew about 700 scientists
  • In 2005, more than 2000

4
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
5
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

6
A Little Debate
  • Video on the sides (approx. 30 min.)

7
Cloning Timeline
  • The Clone Zone

8
Some terms and pictures you may want to know
9
Some of the bigger experiments
  • Cloning is often done with plants
  • 1892 Driesch and his Sea Urchins
  • 1918 vertebrates Spemann and his salamanders

10
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
11
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?

12
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
13
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
14
Success of Somatic Nuclear Transfer Declines With
Older Somatic Donor Cells
  • Interpretation
  • As cells become differentiated, their nuclei
    lose their totipotency

15
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
16
The Technique
17
Clones Not Always Phenotypically Identical
CC A clone
Rainbow The somatic cell donor
18
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?

19
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?

20
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
21
Quit Cloning Around!
  • See the video for yourself!

22
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.

23
Therapeutic Cloning
Heart Cells
24
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
25
Is it Cloning?
  • Play the Game

26
Links
  • Blood Vessel Repair
  • Stem Cell Overview
  • Anti-Aging

27
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.

28
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

29
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

30
Blood Cell Formation
Gradual Restriction in Potency
(uncommitted) (committed)
31
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.

32
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

33
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
34
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.

35
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.

36
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.

37
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
38
When Does Life Begin?
  • Its up to YOU!

Morula?
Sperm? Egg?
Zygote?
1st Heartbeat?
Just Born?
Blastocyst?
39
Chew on This
  • Be informed!
  • Be educated!
  • Be proactive!

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