Lecture 2 Notes: Potency and Genetic Modification

Potency Review and Genetic Modification Strategies

Developmental Potency

• Developmental potency: The ability of a cell to differentiate into different cell types.
• Totipotency:
  • The ability of a single cell to develop into a complete organism, including all embryonic and extra-embryonic tissues (e.g., placenta).
  • Totipotency is present in the very early embryo (2-8 cell stage) and is subsequently lost as cells differentiate.
• Pluripotency:
  • The ability of a single cell to differentiate into any of the three germ layers (ectoderm, mesoderm, and endoderm), and therefore, any cell type in the body.
  • Pluripotent cells can form all tissues of the body but cannot form the placenta.
  • Examples of pluripotent cells:
    • Inner cell mass (ICM) of the blastocyst
    • Embryonic stem cells (ESCs).

Importance of Totipotency

• If a cell is totipotent, genetic changes at this stage will propagate to every single cell of the developed organism, including the germline.

Pluripotency Importance and Testing

• Pluripotency:
  • Self-renewal + developmental potency in a dish.
  • Can be induced by Yamanaka factors (OCT4, SOX2, KLF4, MYC) to form induced pluripotent stem cells (iPSCs).
• Uses of Pluripotent Stem Cells:
  • Transgenic animal production (e.g., knockout mice)
  • Regenerative medicine
  • Disease modeling.

Testing Pluripotency

1. Differentiation in culture:
   • Pluripotent stem cells are cultured in vitro to allow them to differentiate into various somatic cell types.
   • OCT4 is a marker of pluripotency.
2. Teratoma Formation:
   • Pluripotent stem cells are injected into an immunodeficient mouse.
   • If the cells are pluripotent, they will form a teratoma, which is a tumor containing cells from all three germ layers.
3. Blastocyst Injection:
   • Pluripotent stem cells are injected into a host blastocyst.
   • The blastocyst is then implanted into a surrogate mother.
   • The resulting offspring will be a chimera, containing cells from both the host blastocyst and the injected pluripotent stem cells.
4. Tetraploid Complementation:
   • Tetraploid (4N) blastocysts are used as hosts.
   • Tetraploid cells can form a placenta but not an embryo.
   • Therefore, if a viable embryo develops, all of its cells must have come from the donor pluripotent stem cells.

iPSCs and Epiblast Stem Cells

• iPSCs: Induced Pluripotent Stem Cells
  • Generated by reprogramming somatic cells with Yamanaka factors.
• EpiSCs: Epiblast Stem Cells
  • Derived from the epiblast (E6.5 in mice).
• Pluripotency is short-lived.

Gene Modification and Developmentally Potent Cells

• Developmentally potent cells + gene modification = powerful combination.
  • Easiest way to access the germline in mammals.
  • Ensures propagation of the modification.
• Why Gene Modification?
  • Gene 'knockout' illuminates gene function.
  • Transgenics can increase value.
  • Gene editing could cure the disease.

Genetic Modification Strategies

Pronuclear Injection

• DNA is injected into the zygote (pronuclear stage).
• Integration is largely unsuccessful and generally inefficient.
• Examples:
  • Rosita: Argentine cow producing humanized milk via pronuclear injection of 2 human genes.
  • Enviropig: Canadian wonder hog that digests more phosphorus due to a phytase gene (from roundworm) expressed in saliva, making waste more environmentally friendly.

Nuclear Transfer

• Gene modification is done in cultured cells, allowing more complex changes.
• Somatic nucleus is 'reprogrammed' by oocyte factors.
• Example: Dolly the sheep.
• Inefficient: 1-3% in mouse, 20% in cattle.

Embryonic Stem Cells (ESCs)

• Modified ESCs are injected into a host blastocyst to produce a chimera.
• It takes an extra generation to get to the transgenic founder (except for tetraploid complementation).
• Gene modification is easily done in ESCs.
• Multiple rounds of gene targeting can be done.
• Two types of ESCs:

ESC-Based Modification

• Mice with 'humanized' immune systems
  • Total of 2.7 MB added to the mouse genome.
  • 20 different BACs, each made 'markerless'.
  • Inversion/modification of the mouse genome.
  • Antibody drugs = $200 billion USD$ industry.
  • Fastest growing pharmaceutical-type (Lee et al., 2013).

Types of Embryonic Stem Cells (ESCs)

Classical/Primed ESCs (1980s)

• Revolutionary, almost all KO mice have been produced using classical ESCs.
• Allows for multiple rounds of genetic targeting (e.g., mice with humanized immune systems).
• Limitations:
  • Grown with serum.
  • Only works in mice (J1 strain).
  • Primed for differentiation (heterogeneous).

Naïve ESCs (2010s)

• Fully defined media (N2B27).
• '2i' signaling inhibition:
  • Repression of the FGF2 and GSK3B signaling pathways required for post-implantation development.
• Homogenous expression of pluripotency markers (NANOG).
• Works in any mouse strain, rats, and primates (human).

Summary of Germline Entry Methods

Gene Technology Bill

Goals of the Gene Technologies Bill

• Risk-proportionate regulation
• Efficient application and decision-making processes
• A flexible legislative framework able to accommodate future technological and policy developments without frequent amendment
• International alignment, including with key trading partners, to facilitate trade and improve access to new technologies
• Ways to recognize and give effect to the Crown’s obligations under the Treaty of Waitangi

Issues with Current HSNO Act (1996)

• The HSNO Act (1996) is almost 30 years old and out of date.
• Regulation on process, not risk.
• Contradictions regarding definitions.
• Easily outdated.
• Precautionary.
• Effective ban on release of GMOs:
  • Only 3 unconditional releases (all medicines).
  • Handful of field trials.

Public Opinion

• Broad support on medical (and scientific?) use
• Opposition on release:
  • Economic concerns
  • 'Spiritual' concerns