SOMATIC NUCLEAR TRANSFER

Question 1

General introduction about the SNT

Answer 1

What is the SNT and relation with the cloning procedures
1930s: idea from Hans Speman starting from the question “How can a fertilized oocyte develop into a well-organized organism with differentiated cells?” > fantastic experiment
1952: first realization by Briggs and King: Rana pipens, using a nucleus donor a cell from the blastula stage > impossible to obtain the same result (undifferentiated mass) starting from a more differentiated stage
1958: Gurdon repeated the experiments in Xenopus leavis starting from blastula and gastrula stage and obtaining tadpoles from the embryos
1962: Gurdon contested the idea of Briggs and King obtaining tadpoles starting from the nucleus of a differentiated tadpole cell > concept of the nuclear reprogramming

Question 2

Somatic nuclear transfer in mammals

Answer 2

1981: paper reporting the nuclear transplantation in Mus musculus (first mammal)
They use a female mouse (of a specific strain) as donor, inducing the formation of the embryo, they isolated the ICM and took the nucleus from these cells, implanted into a enucleated fertilized oocyte (zygote, difference with the previous experiments in amphibians). Generation of an offspring with characteristics of the strain of the nucleus donor cells.
Importance of the oocyte maturation stage for the success of the nuclear transfer: phase MII

Question 3

Cloning via SNT

Answer 3

Ian Wilmut experiments to obtain the first cloned animal, Dolly the sheep.
First establishment of an embryonic cell line with introduction of the starvation strategy to bring the donor cells in a G0 phase (DNA more accessible to the reprogramming)
Then, they use a somatic cell as nucleus donor from the mammary gland of a 6 years old sheep > first SOMATIC nuclear transfer

Question 4

Mitochondrial DNA

Answer 4

Genetic chimeras: nuclear DNA from the donor + mtDNA from the enucleated oocyte

Question 5

Cross-species SNT

Answer 5

2005: Carp nucleus into a goldfish enucleated oocyte

Question 6

Age of the cloned organisms and problems related to the cloned animals

Question 7

Concept of the therapeutic cloning for gene therapy approaches

Answer 7

Starting from a pre-clinical model of with a genetic defect, somatic cells put in culture, expanded, nucleus transferred into enucleated oocyte, fertilization in vitro, arrival to the blastocyst stage and isolation of ICM cells, corrected by gene therapy approaches and reimplanted in the same animal