Lecture 6 – nuclear reprogramming and cloning Flashcards
Dolly the Sheep and Somatic Cell Nuclear Transfer:
Researcher: John Gurdon et al.
Model Organism: Amphibians.
Technique: Nucleus of somatic cell transferred into oocyte cytoplasm.
Objective: Reverse ‘terminal differentiation’ and form a whole animal.
Transgenic Mice and Sheep (1980s-90s):
Milestone (1980): Transgenic mice created by DNA injection into zygotes.
Treatment Production (1982): Transgenic sheep producing α-l-antitrypsin (AAT) in milk.
Purpose: Cost-effective production of AAT for emphysema treatment.
Transgenic Sheep (1990): Tracy, producing 35g of AAT per liter of milk.
Transgene Suppression and Tracy’s Breeding (1990-97):
Challenge: Transgenes in animals often suppressed.
Approach: Breed Tracy to produce flocks of transgenic sheep.
Outcome: Variable expression levels of AAT in transgenic sheep.
Early Cloning Techniques - Steen Willadsen (1979) and Enucleated Oocytes (1983):
Cloning Method (1979): Sheep cloning by splitting 2-cell embryos.
Cloning Method (1983): Sheep cloning by fusing single cells from 8-cell embryos with enucleated oocytes.
Oocyte State: Arrested in meiosis, diploid, no nuclear membrane, awaiting the second meiotic division.
Regulation: High levels of maturation promoting factor (MPF) control cell cycle progression.
Maturation Promoting Factor (MPF) and Cloning Process:
Components: Protein kinase (cdc2) bound to Ca2+-sensitive cyclin.
Function: Drives the cell through the cell cycle.
Regulation: Sensitive to calcium levels, high calcium inhibits MPF, decreasing its activity.
Early Cloning Attempts - Gurdon’s Work (1950s-60s):
Researcher: John Gurdon et al.
Model Organism: Amphibians.
Technique: Nucleus of somatic cell transferred into oocyte cytoplasm.
Objective: Reverse ‘terminal differentiation’ and form a whole animal.
Fertilization and Calcium Entry:
Event: Calcium enters the oocyte at fertilization.
Source: Released from intracellular stores.
Effect: Destabilizes Maturation Promoting Factor (MPF).
Outcome: Oocyte DNA is released to complete meiosis, forming a haploid pronucleus.
Polar Bodies and Chromosome Elimination:
Function: Serve to eliminate one half of the diploid chromosome set.
Process: Produced as small cellular byproducts during oocyte meiotic division.
Result: Leaves behind a haploid cell.
Zona Pellucida
Definition: Thick transparent membrane surrounding the mammalian ovum.
Timing: Present before implantation.
Male and Female Pronuclei:
Fusion: Male and female pronuclei do not fuse.
Replication: After a while, calcium levels drop, MPF becomes more active, and the two pronuclei replicate independently.
Mitosis and 2-Cell Diploid Embryo:
Process: Pronuclei membranes break down, chromosomes line up, and mitosis completes.
Result: Produces a 2-cell diploid embryo.
Note: Zygote never contains a single diploid nucleus.
Enucleation and Hi-MPF State:
State: Enucleated oocyte cytoplasm is in a Hi-MPF state.
Effect: Donor nucleus breaks down, exposing chromatin to oocyte cytoplasm.
Caution: Donor nucleus should be in G1 or G0 to avoid DNA replication.
Donor Nucleus Preparation:
Requirement: Donor nucleus should be in G1 or G0.
Achieved by: Starving it of mitogenic growth factors for a few days in culture.
Artificial Zygote Activation:
Need: Required when bypassing normal sperm entry/fertilization.
Methods: Chemical activation (strontium ion solution) or electrical activation (small electric shock).
Cloning Strategy:
Process:
Take somatic cells from the donor animal.
Extract chromosomes from a recipient unfertilized oocyte.
Place donor cell nucleus into enucleated oocyte (direct injection/electrofusion).
Activate the oocyte for development.
Transfer to the uterus of a pseudopregnant female.
Complete development in utero.
Oocyte Activation:
Mechanism: Release calcium from intracellular stores.
Effect: Destabilizes MPF, allowing donor nucleus to enter mitosis.
Result: Initiates the first cell division.
Reprogramming and Totipotency:
Reprogramming: Donor DNA decondenses and is reprogrammed by host cytoplasm.
Epigenetic Changes: Loss of epigenetic silencing genes, such as heterochromatic.
Cloning Proof by Wilmut et al.:
Objective: Attempt to make ES cells from sheep.
Outcome: TNT4 cells initially pluripotent but differentiated in culture.
Key Step: Fused differentiated TNT4 cells with enucleated oocytes.
Result: Transplanted into a female, leading to the birth of 5 lambs (e.g., Megan and Morag).
Taffy and Tweed Case:
Finding: Fully differentiated embryonic cells remain potentially totipotent.
Source: Cloned from fetal fibroblast cells from the epidermis of embryos from Welsh black sheep.
Fusion: Fused with enucleated Scottish blackface oocytes.
Outcome: 2 healthy clones born, demonstrating the potential of embryonic fibroblasts for cloning.
Dolly’s Significance:
Milestone: First proof that differentiated cells from adults can be reprogrammed to totipotency.
Technique: Cloned from adult sheep mammary gland cells.
Outcome: Demonstrated the feasibility of cloning animals from fully differentiated cells.
Dolly’s Cloning Process:
Year: 1996
Cells Used: Mammary gland epithelial cells (from udder) cultured for three years.
Fusion: Enucleated oocytes fused 277 times, resulting in 29 blastocysts.
Outcome: Transferred to a sheep, one pregnancy, and birth of Dolly (fertile and genetically identical).
Polly’s Cloning and Transgene Expression:
Year: 1997
Cells Used: Fibroblasts transfected with a transgene encoding human clotting factor IX.
Outcome: 201 embryos, 40 blastocysts, 13 surrogates, 5 pregnancies, and 7 lambs born.
Special Feature: Polly produced factor IX at high levels in milk.
Health Issues in Cloned Animals:
Observations: Cloned animals, including Dolly, reported as unhealthy.
Issues: Respiratory and circulatory problems, weak immune system, liver failure, and premature ageing.
Possible Causes: Epigenetic reprogramming failure, telomere shortening, and retention of donor mutations.
Mice as Cloning Model:
Finding: Serial cloning for many generations in mice can lead to normal-aged animals.
Telomere Observation: Mice telomeres do not shorten with age, contrasting with other species.
Challenges and Ethical Approach:
Proposal: Sophisticated cloning involves making stem cells (ES cells) for therapeutic purposes.
Study Example: Rat injected with dopaminergic neurons (from differentiating ES cells) to alleviate Parkinson’s symptoms.
Objective: Ethically appropriate cloning for therapeutic and reparative applications.
Cloning Applications:
Potential Uses: Study ageing, embryonic development, produce drugs in milk (e.g., cows), protect endangered species, create spare organs without rejection.
