Reproductive Biotech

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Reproductive Biotech
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Superovulation

• treatment of the female with hormones so that
  more eggs are ovulated than normal

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Superovulation

• alter normal level of FSH
• Follicle Stimulating Hormone
• Typical results are ovulation of 10 or more eggs

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Superovulation

• recipient animals must be closely synchronized
  with the donor cow.
• Must be at the same stage in their heat cycle

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EmbryoTransfer

• provides opportunity to introduce genetic
  material into populations of livestock while
  greatly reducing risk of transmission of diseases

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ET Process

• super ovulate donor cow
• synchronize recipients
• breed donor cow (A.I.) when in heat

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ET Process

• flush - remove fertilized eggs from donor before
  7 days gestation
• plant embryo in the recipient animal

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Transfer to recip.

• Non surgical
• common
• deposit embryo in uterus through cervix with AI
  straw and gun

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non surgical

• usually 70-80 success rate

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Surgical

• incision in recip through flank
• tip of uterine horn is exposed
• cut open and embryo is deposited

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Surgical

• recip is sewn shut
• less than 60-65 success rate.

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ET

• now allows for shipping of 500 registered
  holsteins in four sealed flasks in one seat on
  an airplane

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Splitting Embryos

• remove embryo from cow 7 days after conception
• collect unfertilized egg and cut into zona
  pellucida

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Splitting Embryos

• remove contents of unfertilized egg leaving the
  zona intact
• similarly, cut into zona around embryo

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Splitting Embryos

• divide cells in embryo in half using a micro
  surgical blade
• remove 1/2 of the cells and transfer them into
  empty zona

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Splitting Embryos

• seal zona with microjolt
• each 1/2 put into recipient animals

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Cloning

• 6 days after conception, 32 cell embryo is
  removed from donor cow
• recipient egg cell is taken from a slaughterhouse

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Cloning

• recipient cells DNA is removed
• one of the 32 cells is removed from the embryo

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Cloning

• the single cell is inserted into recipient egg
• recipient cell is given a microjolt - fuses
  grafted DNA to plasma membrane

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Cloning

• desired embryo will replace cells that are
  removed providing a continuing supply of DNA to
  be inserted into more recipient cells

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Cloning

• resulting offspring are cloned identical siblings

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Sexing Embryos

• usually done by determining if embryo is a male
• males have Y chromosome
• H-Y antigen can be detected in male cells

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Detection of Y

• break open cells and stain chromosomes to
  identify presence of Y chromosome
• Karyotyping
• must examine at least 10 cells

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Detection of Y

• 70 accurate
• use Y specific probe for ID
• Y specific probe attaches to Y chromosome

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Detection of Y

• can be detected by radioactivity or special light
• 85 accurate

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H-Y Antigen

• make an antibody by injecting H-Y antigen into
  mice
• attach dye onto the antibody for easy detection
  of male embryo

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H-Y Antigen

• add antibody to embryo and if embryo is male it
  will glow
• if it is female it will remain dark

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Advantages

• does not kill cell
• no damage to embryo
• fast - can be few hours or less than one hour old

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Disadvantages

• need high skilled technician
• 80 accurate

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Freezing Embryos

• cooling embryos -5C or 40 F
• can be stored several days
• freezing -196 C or -320 F
• must cool before freezing

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Freezing Embryos

• slowly cooled to -25C at a rate of .5C per minute
  and then plunged into liquid Nitrogen

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Freezing

• cryoprotectants are used to lower the freezing
  point so removal of water can be increased

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Freezing

• Cryoprotectant used in cattle - glycerol
• also help prevent cell membrane from becoming
  brittle
• dilutes salt in cell

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Thawing

• 500 C per minute
• complete is about 20 seconds
• glycerol is removed and embryo is placed in
  successive solutions

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Thawing

• glycerol is removed and embryo is placed in
  successive solutions to re hydrate

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Thawing

• if done too slowly embryo will burst because
  large ice crystals will form
• 70-80 success rate

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Animals

• Genetic Transformation
• two tools
• embryo micromanipultion
• recombinant DNA technology

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Genetic Trans.

• Occurs when new genetic info is introduced into
  animals genome

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Methods

• ES - Embryonic Stem
• EC - Embryonic Carcinoma cells

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ES or EC

• cells harboring the foreign gene are incorporated
  into blastocyst resulting in a mosaic animal

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EC

• potential for tumor formation

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Retrovirus infection

• retro. Carrying desired gene is used to
  transformhost
• close to 100 efficient

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Microinjection

• of several hundred copies of DNA are injected
  into male pronucleus of one or two cell embryos

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Microinjection

• inefficient
• mice - 10-15 are born
• only 25 express new trait

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Screening

• is it transgenic?
• Incorporation of gene
• expression of gene
• germline incorporation

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Agricultural Use

• rabbit
• sheep
• pig
• cow

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The End !! )