ART

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ART

• Assisted Reproductive Techniques

Hunter OReilly, Madonna con Clon 2001
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ART

• Methods or procedures designed to
• Remedy dysfunctional reproduction
• Increase reproductive success
• Expand knowledge in fields of genetics and
  reproductive physiology

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ART

• Techniques include
• IVF - in vitro fertilization
• ICSI - intracytoplasmic sperm injections
• PGD - preimplantation genetic diagnosis
• GIFT - gamete intrafallopian transfer
• ZIFT - zygote intrafallopian transfer
• Sex determination
• NT - nuclear transfer

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In Vitro Fertilization

• in vitro in glass
• Involving the insemination of an embryo that was
  previously fertilized in culture into a female
  recipient.

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In Vitro Fertilization

• Methodology
• 1. Superovulation harvest of oocytes
• Kept at 37C in balanced salt solution
• Cyropreserved
• 2. Insemination of oocyte with sperm cell
• Sperm is allowed to naturally swim to
• oocyte and join for ferilization

Courtesy of http//www.gfmer.ch/Livres /FIV_atlas/
FIV_Images.htm
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In Vitro Fertilization

• Methodology
• 3. Culture of embryos
• Micromanipulation for biopsy occurs here
• Grown to the blastocyst stage
• 4. Embryo transfer
• Transfer of embryo from culture to implantation
  into female recipients

Courtesy of http//www.reproductivegenetics.com
Courtesy of http//www.shadygrovefertility.com
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In Vitro Fertilization

• First successful attempt to conceive a human
  child in 1978.
• Louise Brown
• British baby girl, born July 25, 1978
• Today is 26 and lives in Bristol, England
• Is a postal worker and recently married
• Since then, 1 million babies born via IVF
• Spawning 115,000 births in U.S. alone
• Cost 7-10,000
• Vary in donor age and reproductive complication

Courtesy of http//www.babycenter.com/general/9811
.html
Courtesy of http//news.bbc.co.uk/1/hi/health/3091
241.stm
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In Vitro Fertilization

• Advantages
• Correct oocyte fertilized to correct sperm
• Embryo biopsy to assess genetic status
• Remedies the issue of infertility affecting 6.1
  million people (male and female)
• Increase reproductive potential of genetically
  superior animals

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Intracytoplasmic sperm injections

• aka. Microinsemination
• Process by which a single sperm cell is injected
  into an oocyte
• Regardless of quality or level of maturity
• Non-motile sperm cells can be used
• In case of male infertility due to
• Low motility, viability, and quantity
• Abnormally shaped

Courtesy of http//www.ivf.com/insem.html
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Intracytoplasmic Sperm Injections

• Procedure
• 1. Gamete cells harvested and prepared as in IVF
• 2. Sperm cells cultured in medium and oocytes
  exposed to hyaluronidase
• Enzyme destroys cumulus layer to expose oocyte
  for ICSI
• 3. Micropipette equipped with gentle vacuum holds
  egg in place
• 4. Hollow needle containing sharp edge is used
• To draw up a single sperm cell
• Penetrate the zona pellucida of the oocyte
• Deposit sperm cell into cytoplasm of oocyte

Courtesy of http//www.ivf.com/ivf_icsi.html
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Intracytoplasmic Sperm Injections

• Procedure (contd)
• 6. Embryo culture observation
• Formation of 2 pronuclei
• Signaling successful fertilization
• 7. Embryo culture and maintenance
• Only healthiest embryos selected
• Embryo biopsy or screening can occur at this
  time
• 8. Embryo transfer
• Stage of embryo development dependent upon
  species
• Livestock 5-6 day cultures

Courtesy of http//www.ivfmississippi.com/
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Preimplantation Genetic
Diagnosis

• Purpose
• Screening of embryos for genetic mutations,
  chromosomal errors, sex selection and
  determination for sex-linked chromosomal
  abnormalities.
• Procedure
• 1. Removal of 1st polar body from oocyte
• 2. Removal of one of the cells in developing
• embryo in the 6-12 cell stage
• 3. Removal of cells from trophectoderm

Courtesy of http//www.ivf.com/insem.html
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Preimplantation Genetic Diagnosis

• Analysis for genetic disorders
• FISH (Fluorescence in situ hybridization)
• Fluorescent probes for specific chromosomes
• Binding will cause illumination and indicate
  presence of chromosome of interest
• PCR (Polymerase Chain Reaction)
• Amplification of specific DNA fragment or
  sequence of genetic material within the cell
• Results obtained within a day
• Risk of harm during procedure exists

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Alternative ET Methods

• GIFT
• Gamete intrafallopian transfer
• Both sperm and oocyte are placed inside female
  fallopian tubes
• Natural fertilization
• ZIFT
• Zygote intrafallopian transfer
• IVF embryo inserted into females fallopian tubes
• Differs from standard IVF, not implanted in
  uterus

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Sex Determination (sperm embryo)

• In mammalian species, the male determine sex or
  gender of offspring via sperm cells (X or Y).
• Selection between X and Y sperm cells
• PGD development of probes or localization of
  sex chromosome
• Microsort sperm separation separation of X and
  Y chromosome bearing sperm
• X chromosome negatively charged, higher mass
  than Y bearing sperm
• Y chromosome positively charged, lower mass,
  cell surface antigen
• Embryo or fetus prenatal testing
• Ultrasound scanning (non-invasive)
• Amniocentesis CVS (invasive)

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Sperm Sex Sorting

• Various procedures
• Electrophoresis
• Sample in buffered media exposed to anode/cathode
• Sperm cells move according to cell surface charge
• Sedimentation
• Sample allowed to settle in density gradient
  media
• Equilibrium point sperms specific gravity
• Immunological
• Y-bearing sperm cell surface antigen, highly
  conserved
• Developed antibody either binds to or destroys
  Y-sperm

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Sperm Sex Sorting

• Combo Procedure
• Convection counter streaming-galavanization
• Electrophoresis Sedimentation
• DNA content X gt Y
• Dr. Larry Johnson, USDA Beltsville

Courtesy of XY Inc., http//www.xyinc.com/sexselec
t/moflo.php
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Sex Determination

• Screening of sex-linked chromosome abnormalities
• ie. Duchennes muscular dystrophy or hemophilia
• Afflicting only males, found on Y chromosome
• Only female embryos selected for implantation
• The skinny on the old wives tale
• Intercourse timing and ovulation induction has
  not shown to modulate sex ratio.

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Nuclear Transfer

• Definition
• Involves the insertion of a nucleus from a
  differentiated somatic cell into an un-nucleated
  oocyte.
• Nucleus of the somatic (adult) cell is
  reprogrammed into a pleuropotent cell by the
  cytoplasm of the oocyte.
• Functions on the principle that the cytoplasm
  from the oocyte contains factors that can
  reactivate all genes in any somatic or
  differentiated nucleus converting it back into a
  stem cell.
• Can also be accomplished with fusing and entire
  diploid cell into an oocyte and switched on with
  an electrical impulse
• Reconstructed embryo

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Nuclear Transfer

• History
• Techniques began in 1950 for the study of frog
  development.
• Spread into livestock cloning in 1980s.
• Successful attempts
• Megan and Morag created in 1995
• Lambs created from week-old embryo cultures
• Marked 1st time an animal was derived from
  cultured cells
• Roslin Inst. PPL Therapeutics
• Created 1st animal from a somatic (adult)
  cell in 1996
• Birth of Dolly in 1997
• Dolly has a baby lamb named Bonnie

Courtesy of http//www.islamset.com/healnews/cloni
ng/wilmut.html
Courtesy of http//www.roslin.ac.uk/public/cloning
.html
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Nuclear Transfer

• Cloning of Dolly
• Nucleus of mammary gland cell of 6-yr old sheep
  was transferred into an oocyte.
• Normal development of a 5-6 day culture was
  placed inside a female recipient.
• Proved that somatic cells are not fixed in their
  roles.
• Received the Science Breakthrough of the Year

Courtesy of http//www.roslin.ac.uk/public/cloning
.html
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Jury is still out on Dollys Problems

• Premature aging
• Dolly was confirmed to be chromosomally older
  than her birth date according to her telomere
  length.
• One of the obstacles in cloning.
• Age of cell harboring nucleus to be transferred
  remains and continues to age from this point
  despite new cytoplasm.
• As cells divide and differentiate, they age and
  have a limited lifespan.
• DNA stretches and chromosome ends (telomeres)
  fray and shorten.
• DNA is rendered susceptible to errors and
  mutations.

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Cloning via Nuclear Transfer

• In 1963, Chinese embryologist Tong Dizhou, cloned
  a carp. Obscured in a Chinese science journal
  never translated to English.
• Success in mice, cattle, sheep, goats, and pigs,
  rhesus monkey, garu, cat, rabbit, mule, deer,
  horse, rat, and fruitfly.
• Success rates leading to live births are very low
  in all species.
• Success rate of 1.
• Differences exist in early embryo development.
• Death occurring in late pregnancy improper
  placental development.
• Death occurring right after birth neonate much
  larger than normal.
• High incidence of genetic abnormality.

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Cloning Cats
Cells from Rainbow were cloned to get
Cc. Surprisingly the cats dont look or act
alike. Rainbow is a typical calico with
splotches of brown, tan and gold on white. Cc
has a striped gray coat over white. Rainbow is
reserved. Cc is curious and playful. Rainbow is
chunky. Cc is sleek.
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What else has AM cloned?
Second Addition, a Boer goat was born in
2001 Dewey, the world's first deer clone, was
born May 23, 2003.
Second Chance, first cloned bull was born in
1999.
First piglets cloned were born August 12, 2001.
86 Squared, first animal cloned for disease
resistance, was born in November 2000.
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And still more clones coming
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Cloning Abnormalities

• Chromatin structure
• Somatic cells vary compared to gamete cells.
• Transferred nucleus must be reprogrammed
  immediately during activation of reconstructed
  embryo.
• Abnormalities are due to
• Improper methylation of DNA and inappropriate
  reconfiguration of chromatin following
  fertilization.
• Causing deregulation of expression of genes.
• Normal embryo development failure.

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Limitations of Nuclear Transfer

• Success requires an intact nucleus with
  functional chromosomes.
• Most preserved species have lost their DNA
  integrity
• Fragmented DNA
• Complete genome is destructed
• As of yet, no hope for the rebirth of the
  Tasmanian tiger or Siberian mammoth.
• Farm animal production and cloning
• Integration into breeding program to profit.
• Care taken on genetic diversity preservation.

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?? Questions??
Hunter OReilly, Unique Clones 2001