Nuclear Transfer & Reproductive Cloning Flashcards
What are two ethical issues raised by nuclear transfer and cloning?
- It isn’t risk free. Dolly died at half the appropriate age, the cloned guar died after birth, and cloned mice have been found with genetic abnormalities.
- In the absence of implantation, a genetically constructed embryo (based on a somatic nucleus and unfertilized egg) cannot become human.
- Spare parts argument?
What’s a monozygotic twin?
A naturally occurring two-celled embryo separates into two distinct humans.
What is embryo splitting?
An embryo’s cells are split into two blastomeres at an early stage (similar to monozygotic twins).
When was the first cloning by embryo splitting performed and by whom?
- Hans Spemann (German)
2. He used salamanders and then began to toy with transferring somatic cell nuclei into enucleated (?) oocytes
When was the first cloning and who performed it?
- Briggs and King (in the 1950’s)
2. Injected adult frog kidney carcinoma cell nuclei into enucleated oocytes of northern leopard frogs
When was the first plant cloned and by whom?
- In the 1950’s by Steward from Cornell Uni
2. Cloned single cells in vitro of carrots and then reprogramed differentiated cells (later they used other plants)
What are three important dates and occurrences re:the cloning of sheep?
- 1995 Wilmut and Campbell used differentiated embryo cells to clone Megan and Morag
- In 1996 Dolly was cloned from two adult cells although there are discrepancies.
- In 1997 Wilmut and Campbell injected in Polly the human factor 9 gene (clotting for hemophilia) so she is a transgenetic animal.
What are the steps for nuclear transfer?
- A donor cell is selected from an adult organism.
- Nucleus of recipient cell (unfertilized oocyte) is selected.
- Nucleus of donor cell is transferred into the recipient cell.
- New cell is activated to divide as if it were fertilized.
(this was used for Dolly)
What did Dolly’s cloning disprove?
- The nucleus from a differentiated cell cannot reverse into pre-zygotic conditions (adult somatic cells RETAIN genetic totipotency!).
- Germ cells lose their special status?
What needs to be synchronized for proper ploidy during nuclear transfer?
The cell cycle of the selected nucleus with the selected recipient cytoplast
What does the cytoplasm contain which affects cell cycle?
Protein kinases and other proteins
What factors affect the policy of cloned cells?
- The donor cell’s current growth cycle phase
- Ditto for recipient cell
- When fusion occurs
- Note: the actual mechanism for somatic cell nucleus in ooplasm is unknown.
Gene remodeling and DNA expression occurs after what?
Nuclear transfer
What kinds of cells are used for donor cells?
- mammary epithelium
- cumulus cell (donor of first cloned mouse Cumulina)
- primary fibroblast
- B-cell
- T-cell
- later pre-implantation fetal cells
- later pre-implantation embryo cells
What cells are more efficient in creating clones and why?
embryonic stem cell nuclei are more efficient than granolas cells or lymphocytes (probably because they are undifferentiated, similarly to gametes)
At what stages of the cell cycle has cloning been achieved?
- Dolly G0
2. Also at G1, M, and G2
IVF technicians do what to achieve nuclear transfer images?
- Remove DNA from recipient and stain with Hoesche dye
- Remove PB
- Aspirate spindle from an unfertilized metaphase oocyte and check by fluorescent microscopy
- Insert donor cell under zona
- Electrofuse the recipient cell with donor cell and culture
Why is the PB removed from the DNA of a recipient cell during nuclear transfer?
LOOK UP!
What are some future applications of therapeutic cloning?
Use cloned embryos to generate new cells/tissues/etc. to cure things like diabetes, Parkinson’s disease, Alzheimer’s, etc. (no risk of rejection)
What are some problems with cloning?
- Low efficiency (1/227 nuclear transfers for Dolly)
- Health problems of cloned animals (large fetus syndrome, obesity, pneumonia, liver necrosis, prolonged gestation, leukemia, lung cancer, early deaths, shortened telomeres, malfunctioning immune systems)
- Possibly caused by inappropriate genetic reprogramming after transfer
What was the 1993 Hall & Stillman experiment?
- Split polyploid embryos
- Aimed to increase pregnancy chances and/or make MZ twins
- Out of 48 embryos, none lasted after day 6
- Became controversial b/c of dying of human embryos as result of experiment
What are the three origins of monozygotic twinning?
- Splitting before trophoblast formation (each twin has its own amnion and chorion).
- Splitting between trophoblast and amnion formation (each twin has its own amnion but share a chorion).
- Splitting after amnion formation (both twins in same amnionic sac and share chorion).
Why does ART increase the frequency of monozygotic (and DZ) twinning?
Possibly because the zona is hardened and this makes a pinching off of the inner cell mass and trophectoderm (it’s verified in certain cases).
What four risks are there for twins?
- Premature delivery
- Birth defects
- Cord strangulation (especialy in monoamniotic twins)
- TTTS (twin-to-twin-transfer syndrome) where identical twins have abnormal connections of placental blood vessels and the flow from one twin to another is imbalanced, which seriously risks survival and health of both)