Lecture 6 – nuclear reprogramming and cloning

Question 1

Dolly the Sheep and Somatic Cell Nuclear Transfer:

Answer 1

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.

Question 2

Transgenic Mice and Sheep (1980s-90s):

Answer 2

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.

Question 3

Transgene Suppression and Tracy’s Breeding (1990-97):

Answer 3

Challenge: Transgenes in animals often suppressed.
Approach: Breed Tracy to produce flocks of transgenic sheep.
Outcome: Variable expression levels of AAT in transgenic sheep.

Question 4

Early Cloning Techniques - Steen Willadsen (1979) and Enucleated Oocytes (1983):

Answer 4

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.

Question 5

Maturation Promoting Factor (MPF) and Cloning Process:

Answer 5

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.

Question 6

Early Cloning Attempts - Gurdon’s Work (1950s-60s):

Answer 6

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.

Question 7

Fertilization and Calcium Entry:

Answer 7

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.

Question 8

Polar Bodies and Chromosome Elimination:

Answer 8

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.

Question 9

Zona Pellucida

Answer 9

Definition: Thick transparent membrane surrounding the mammalian ovum.
Timing: Present before implantation.

Question 10

Male and Female Pronuclei:

Answer 10

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.

Question 11

Mitosis and 2-Cell Diploid Embryo:

Answer 11

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.

Question 12

Enucleation and Hi-MPF State:

Answer 12

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.

Question 13

Donor Nucleus Preparation:

Answer 13

Requirement: Donor nucleus should be in G1 or G0.
Achieved by: Starving it of mitogenic growth factors for a few days in culture.

Question 14

Artificial Zygote Activation:

Answer 14

Need: Required when bypassing normal sperm entry/fertilization.
Methods: Chemical activation (strontium ion solution) or electrical activation (small electric shock).

Question 15

Cloning Strategy:

Answer 15

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.

Question 16

Oocyte Activation:

Answer 16

Mechanism: Release calcium from intracellular stores.
Effect: Destabilizes MPF, allowing donor nucleus to enter mitosis.
Result: Initiates the first cell division.

Question 17

Reprogramming and Totipotency:

Answer 17

Reprogramming: Donor DNA decondenses and is reprogrammed by host cytoplasm.
Epigenetic Changes: Loss of epigenetic silencing genes, such as heterochromatic.

Question 18

Cloning Proof by Wilmut et al.:

Answer 18

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).

Question 19

Taffy and Tweed Case:

Answer 19

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.

Question 20

Dolly’s Significance:

Answer 20

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.

Question 21

Dolly’s Cloning Process:

Answer 21

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).

Question 22

Polly’s Cloning and Transgene Expression:

Answer 22

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.

Question 23

Health Issues in Cloned Animals:

Answer 23

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.

Question 24

Mice as Cloning Model:

Answer 24

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.

Question 25

Challenges and Ethical Approach:

Answer 25

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.

Question 26

Cloning Applications:

Answer 26

Potential Uses: Study ageing, embryonic development, produce drugs in milk (e.g., cows), protect endangered species, create spare organs without rejection.