PP26 Cloning

1
PP26Cloning Assisted Reproductive Technologies
Chapter 13 pg 296-314
ANS 3319Dr. Michael J. FieldsUniversity of
Florida
2
Cloning by Nuclear Transplantation

• Differentiated cells (Dolly)
• Somatic Cell Nuclear Transfer (SCNT), or
• embryonic stem cells
• Transfer into enucleated oocyte (intact ZP).
• Embryo transfer into Universal Recipient.

3
CloningUses in Agriculture Biomedical
Sciences

• Cloning.
• Differentiation.
• Genetic Engineering.
• Transplantation.

4
Cloning by Nuclear Transplantation

• Transfer into enucleated oocyte.
• Each cell (blastomere) is identical but may be
  average.
• Expensive.
• Calves born weak and 20 larger (LOS).
• 10 of transferred cloned embryos make it to
  term.
• 75 of clones die during Peri-Implantation.
• Calves can be genetically abnormal, but in some
  cases appear to be normal (Dolly).
• Problem in placenta or In-Vitro incubation
  (media) IGF-1.
• Calves develop hydroallantois of the placenta.

5
Totipotent

• Embryonic cells having unlimited capability to
  differentiate into all membranes and tissues.
• Undifferentiated in cow up to 32 cell stage.
• Rat 2 cells.
• 30 of cells are totipotent in 200 cell embryo.

6
Pluripotent

• Capable of giving rise to all 3 embryonic tissue
  layers.
• Inner Cell Mass cells give rise to specialized
  tissue but not to a placenta (trophblasts).
• Blood stem cells white, red and platelets.
• Blood stem cells grow into nerve cells.
• Nerve stem cells grow into blood cells.
• Depends on the environment of the cells.

7
Cloning Differentiation

• Cells can be induced to differentiate into any
  cell type given the appropriate environment.
• Can be used in studies looking at factors
  effecting embryonic development including gene
  effects.

8
CloningGenetic Engineering

• Mix embryonic stem cells and targeting vector
  (gene of interest foreign DNA).
• Electroporate (electrical charge) cells and
  foreign DNA.
• .001-.0001 recombinancy rate (very low).
• Select for cells that took up the foreign gene.

9
CloningGenetic Engineering

• Gene targeting altering function of a gene via
  gene transfer.

• Knockout disrupt gene function.
• Knockin replace gene with another.

10
Recombinant Gene Transfer Technique

• Low frequency of success
• Mice 30.
• Farm animals 1
• Plasmid and Pro-Nuclei do not always
  integrate.
• Random uptake.
• Disparities in expression due to different
  numbers of gene copies.

11
Stem Cells

• Transplant Spermatogonia (2N) into testes.
• Produce sperm.
• Men undergoing therapy.
• Remove stem cells and re-implant
  (auto-transplantation).
• Prize stallions.
• Transfer cells into lesser animals.
• Bengal tiger into house cat.
• Preservation of endangered species.
• Insert foreign gene into stem cells and insert
  stem cell into embryo.

12
Organ Donor Pigs

• Xenotransplantation.
• Express human antigens in pig organs so organ
  will not be rejected.

13
DNA Typing

• PCR a gene of interest.
• To confirm insertion.
• Embryo or offspring black (polled homogenous).
• However, many traits may be influenced by a
  number of genes.
• Find a marker for the genes of interest.
• Very little sample needed.
• Amplify the gene.

14
Genetically Engineered LivestockTransgenics

• Functional gene incorporated as a foreign gene
  into genome.
• Insert Growth Hormone DNA into pigs.
• Insert human albumin into cows.
• Insert human Tissue Plasminogen Activatior into
  goats and recover in milk.
• Improve wool 5 - 10.
• Pharming pharmaceutical/vaccine production.
• High insulin production in milk of dairy cows

15
Genetically Engineered LivestockTransgenics

• Increased heat tolerance.
• Disease resistance.
• Higher nutritional efficiency.
• Increased reproductive capacity.
• Modification of muscle mass.
• Better milk production.

16
Transgenic

• Gene knockouts (KO) gene no longer expressed.
• Oxytocin KO (mammary gland).
• Milk release inhibited pups die.
• PGF2 a KO CL does not regress fetuses die.
• Relaxin KO No nipple formation pups die
• Estrogen Receptor KO
• Growth, but no reproduction.
• Calpastatin correlates with meat tenderness.
• IGF-1 correlates with weight gains.
• E2 receptor/additional copy results in additional
  pig/litter.

17
Gene Therapy

• Introduce DNA into human cells.
• Hemoglobulin gene into Sickle Cell Anemia
  patient.
• Tumor Necrosis Factor (TNF) shrinks tumors.
• SCID (Adenosine Deaminase Deficiency) gene
  inserted into lymphocytes.
• Cystic Fibrosis.

18
Assisted Reproductive Technologies (ART)In Vitro
Fertilization/Embryo Transfer (IVF/ET).

• Ovarian stimulation.
• Ultrasound guided recovery of oocyte.
• Oocyte and sperm.
• Embryo placed in uterus or oviduct via catheter.
• Only ART recommended for tubal infertility.

19
Assisted Reproductive Technologies (ART) Gamete
Intrafallopian Transfer (GIFT)

• Ultrasound guided recovery of oocyte.
• Place oocyte and Sperm into oviduct.
• Requires laparotomy.
• Highest rate of success (35 optimal).

20
Assisted Reproductive Technologies (ART) Zygote
Intrafallopian Transfer (ZIFT)

• Ultrasound guided recovery of oocyte.
• In Vitro Fertilization.
• Best in the case of reduced male fertility.
• Embryo/Zygote placed back in oviduct.
• Requires laparotomy.

21
Assisted Reproductive Technologies (ART)Male

• Zone Pellucida drilling/enzymatic digestion.
• Subzonal sperm injection (SUZI).
• Intracytoplasmic sperm injection (ICSI).
• Insert DNA of haploid sperm.
• Can clone with diploid primary spermatocyte.

22
Assisted Reproductive Technologies (ART)
Intrauterine Insemination/Superovulation

• Embryo biopsy.
• Sex determination by PCR of Y chromosome DNA.