Nuclear Transfer & Reproductive Cloning

Question 1

What are two ethical issues raised by nuclear transfer and cloning?

Answer 1

1. 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.
2. In the absence of implantation, a genetically constructed embryo (based on a somatic nucleus and unfertilized egg) cannot become human.
3. Spare parts argument?

Question 2

What’s a monozygotic twin?

Answer 2

A naturally occurring two-celled embryo separates into two distinct humans.

Question 3

What is embryo splitting?

Answer 3

An embryo’s cells are split into two blastomeres at an early stage (similar to monozygotic twins).

Question 4

When was the first cloning by embryo splitting performed and by whom?

Answer 4

1. Hans Spemann (German)

2. He used salamanders and then began to toy with transferring somatic cell nuclei into enucleated (?) oocytes

Question 5

When was the first cloning and who performed it?

Answer 5

1. Briggs and King (in the 1950’s)

2. Injected adult frog kidney carcinoma cell nuclei into enucleated oocytes of northern leopard frogs

Question 6

When was the first plant cloned and by whom?

Answer 6

1. 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)

Question 7

What are three important dates and occurrences re:the cloning of sheep?

Answer 7

1. 1995 Wilmut and Campbell used differentiated embryo cells to clone Megan and Morag
2. In 1996 Dolly was cloned from two adult cells although there are discrepancies.
3. In 1997 Wilmut and Campbell injected in Polly the human factor 9 gene (clotting for hemophilia) so she is a transgenetic animal.

Question 8

What are the steps for nuclear transfer?

Answer 8

1. A donor cell is selected from an adult organism.
2. Nucleus of recipient cell (unfertilized oocyte) is selected.
3. Nucleus of donor cell is transferred into the recipient cell.
4. New cell is activated to divide as if it were fertilized.
   (this was used for Dolly)

Question 9

What did Dolly’s cloning disprove?

Answer 9

1. The nucleus from a differentiated cell cannot reverse into pre-zygotic conditions (adult somatic cells RETAIN genetic totipotency!).
2. Germ cells lose their special status?

Question 10

What needs to be synchronized for proper ploidy during nuclear transfer?

Answer 10

The cell cycle of the selected nucleus with the selected recipient cytoplast

Question 11

What does the cytoplasm contain which affects cell cycle?

Answer 11

Protein kinases and other proteins

Question 12

What factors affect the policy of cloned cells?

Answer 12

1. The donor cell’s current growth cycle phase
2. Ditto for recipient cell
3. When fusion occurs
4. Note: the actual mechanism for somatic cell nucleus in ooplasm is unknown.

Question 13

Gene remodeling and DNA expression occurs after what?

Answer 13

Nuclear transfer

Question 14

What kinds of cells are used for donor cells?

Answer 14

1. mammary epithelium
2. cumulus cell (donor of first cloned mouse Cumulina)
3. primary fibroblast
4. B-cell
5. T-cell
6. later pre-implantation fetal cells
7. later pre-implantation embryo cells

Question 15

What cells are more efficient in creating clones and why?

Answer 15

embryonic stem cell nuclei are more efficient than granolas cells or lymphocytes (probably because they are undifferentiated, similarly to gametes)

Question 16

At what stages of the cell cycle has cloning been achieved?

Answer 16

1. Dolly G0

2. Also at G1, M, and G2

Question 17

IVF technicians do what to achieve nuclear transfer images?

Answer 17

1. Remove DNA from recipient and stain with Hoesche dye
2. Remove PB
3. Aspirate spindle from an unfertilized metaphase oocyte and check by fluorescent microscopy
4. Insert donor cell under zona
5. Electrofuse the recipient cell with donor cell and culture

Question 18

Why is the PB removed from the DNA of a recipient cell during nuclear transfer?

Answer 18

LOOK UP!

Question 19

What are some future applications of therapeutic cloning?

Answer 19

Use cloned embryos to generate new cells/tissues/etc. to cure things like diabetes, Parkinson’s disease, Alzheimer’s, etc. (no risk of rejection)

Question 20

What are some problems with cloning?

Answer 20

1. Low efficiency (1/227 nuclear transfers for Dolly)
2. Health problems of cloned animals (large fetus syndrome, obesity, pneumonia, liver necrosis, prolonged gestation, leukemia, lung cancer, early deaths, shortened telomeres, malfunctioning immune systems)
3. Possibly caused by inappropriate genetic reprogramming after transfer

Question 21

What was the 1993 Hall & Stillman experiment?

Answer 21

1. Split polyploid embryos
2. Aimed to increase pregnancy chances and/or make MZ twins
3. Out of 48 embryos, none lasted after day 6
4. Became controversial b/c of dying of human embryos as result of experiment

Question 22

What are the three origins of monozygotic twinning?

Answer 22

1. Splitting before trophoblast formation (each twin has its own amnion and chorion).
2. Splitting between trophoblast and amnion formation (each twin has its own amnion but share a chorion).
3. Splitting after amnion formation (both twins in same amnionic sac and share chorion).

Question 23

Why does ART increase the frequency of monozygotic (and DZ) twinning?

Answer 23

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).

Question 24

What four risks are there for twins?

Answer 24

1. Premature delivery
2. Birth defects
3. Cord strangulation (especialy in monoamniotic twins)
4. 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)

Question 25

What two types of micromanipulations are used to improve embryo quality?

Answer 25

1. Nuclear transfer

2. Cytoplasmic transfer

Question 26

What four steps are used for nuclear transfer in mice?

Answer 26

1. Use large pipette to aspirate germinal vesicle from oocyte after trading with cytochalasin B (which inhibits polymerization of microtubules and actin that stop formation of polar bodies in recipient).
2. Transfer germinal vesicle to enucleated oocyte.
3. Allow to mature to metaphase II (which takes 16 hours for mice) and fertilize (or activate).
4. Transfer maternal pronuclei to in vivo fertilized oocyte (after removing the female pronuclei) to obtain offspring.

Question 27

What five steps are used for cytoplasmic transfer?

Answer 27

1. Aspirate donor oocyte cytoplasm without messing up the spindle.
2. Remove the recipient PB (what’s that??)
3. Insert the donor’s cytoplasm under the zona.
4. Electrofuse the two parts of the cytoplasm.
5. Fertilize using ICSI
   * **It may improve the quality of oocytes to the detriment of the mitochondria

Question 28

Why may nuclear or cytoplasmic transfer be used?

Answer 28

It is claimed that it improves pregnanycy rates in reproductively aged women (especially after repeated IVF failure due to mitochondrial disease) assuming the quality of the untested donor oocyte.

Question 29

What are six important things to know about nuclear/cytoplasmic transfer today?

Answer 29

1. Associated with mitochondrial heteroplasmy (the presence of more than one person’s mitochondria) which is harmless and occurs in both nuclear as well as cytoplasmic transfers.
2. Possibility of nucleo-cytoplasmic incompatibility.
3. Two fetuses were born with 45X which is worrisome because this is out of a total of only 30 successful cytoplasmic transfer babies to date.
4. FDA license is needed in the USA which prohibits the technique from most labs.
5. In vitro maturation limits the nuclear transfer’s effectiveness which is why the double nuclear transfer is done.