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Two predominant methods of nuclear transfer ; based on different cytoplast used (Collas & Barnes, 1994 ; Ritchie & Campbell, 1995)
a. Use metaphase II oocytes as cytoplast / recipient cell
b. Use zygotes as cytoplast
Blastomeres as source of donor cells. The donor cells forced into the Gap Zero, or G0 cell stage, a dormant phase, which causes the cell to shut down but not
die.
A. Metaphase II (M2) Oocytes Cytoplast
i. Incubate M2 oocyte in medium
containing
microfilament
inhibitor
cytochalasin
D
&
DNA-specific
fluorochrome
Hoechst
3332
iii. using enucleation pipette, the karyoplast is inserted into the hole at zona
pellucida
iv. karyoplast is expelled into the perivitelline space contact with the cell
membrane of enucleated oocyte/zygote (cytoplast)
v. Cell fusion : induced by direct current electric pulse at 90
contact activate the oocyte/zygote
to the plane of
vi. The activated egg cell/zygote is then quickly to begin forming an embryo.
Purpose I. To harvest stem cells Embryonic stem cells (ESCs)
The egg containing the transferred nucleus is encouraged to divide until it
reached the blastocyst stage,
The cells of the inner cell mass are removed and cultured. These are known
as embryonic stem cells, or ESC's.).
Use for therapeutic cloning
Cell Cycle: Oocytes Fertilized egg two pronuclei zygote Blastomeres (2-cell)
-
ii.
III. There are two techniques which are the variation of SCNT
A. Roslin Technique cloning of Dolly (lamb)
Developed by Ian Wilmut and Keith Campbell from Roslin institute, Uni Edinburgh in 1996
Rosalin method, they used donor nucleus from adult cells (mammary gland cells) and cultured the cells (Issue 2)
MII chromosome
Important principle: Synchronize the cell cycles of the donor cell and recipient cell (Issue 1) to ensure the
nucleus is in phase for development of embryo with the right DNA chromosomal content
Significance:
1. Dolly, the first animal cloned from a cell
taken from cultured adult cells
2. enhance the production of GM animals
Increase livestock, as animal breeding
strategies
3. open the field of reprogramming of
nucleus rapid expansion of stem cell
biology & stem cell therapies
Limiitation: cloning extinct species?
- require functional chromosomes (donor)
- require appropriate supply of oocytes &
Methods:
1. 2N Donor cells from udder cells of Finn Dorset sheep (white face) and culture the donor cells in vitro
2. The cells were taken and starved in deprived nutrient media, only enough for cells to live force cells to
be dormant and paused at G0 phase
3. The M2 oocyte (low MPF level) as universal recipient from black face ewe. The nucleus (M2
chromosome & polar body) were removed (enucleation)
4. The donor cells (karyoplast) were in contact, near the enucleated oocyte
5. Use electrical pulse to fuse the donor cell & enucleated oocyte (mimicking fertilization by sperm not so
correct as not all activated oocyte embryo)
6. Current use for fuse = current use to activate oocyte (oocyte is activated immediately after fusion)
7. Activate the oocyte for zygote development reconstructed embryo Culture until reach morula /
blastocyst implant in the uterus of foster mother (black face ewe)
8. Embryo developed Dolly (Finn dorset sheep white face)