Cloning and Genomic Equivalence

Question 1

What happens as a cell starts do differentiate?

Answer 1

Gradual restriction in potency

Question 2

Why is there a restriction in potency?

Answer 2

• Gene loss

- Differential gene activity (largely this is the case)

Question 3

What experiment provides evidence for differential gene activity in potency?

Answer 3

Lens regneration in the newt

Question 4

Process of lens regeneration in the newt experiment

Answer 4

1. Remove lens
2. Proliferation of cells in dorsal iris
3. Synthesis of crystallin proteins (found in lens)
   Iris cells nuclei have genes for crystallin but wouldn’t normally express it

Question 5

What proteins are expressed in the iris to regenerate the lens in newts?

Answer 5

Crystallin proteins

Question 6

What is a heterokaryon?

Answer 6

A heterokaryon is a multinucleate cell that contains genetically different nuclei

Question 7

Human liver cell transplanted into rat muscle cell

Answer 7

Grow these cells together and treat with chemicals so that the membranes fuse together –> heterokaryon
- assay for human liver-specific gene expression and muscle-specific gene expression

Question 8

What did results of a human liver cell transplanted into a rat muscle cell show?

Answer 8

Liver gene silenced and human muscle gene switched on. Gene wasn’t lost but cytoplasm of muscle cell induced change

Question 9

Nucleus from Xenopus kidney cells into Pleurodeles oocyte (newt)

Answer 9

Kidney-specfific genes were silenced. Expressed oocyte-specific genes

Question 10

What does the fact that differentiated Xenopus kidney cells expressed oocyte-specific genes when transferred?

Answer 10

Genetic info is not lost after cells differentiate

Question 11

Conclusions about gene expression

Answer 11

• Gene expression in nuclei from differentiated cells can be changed under special circumstances
• Gene expression can be controlled by cytoplasmic factors
• Genes are not lost when cells differentiate

Question 12

Testing nuclear potency 1: Nuclear transfer in Rana (frog)

Answer 12

Enucleated egg cell and nucleus from cell in blastula stage of an embryo
Shows cells lose potency but nuclei retain it

Question 13

Testing nuclear potency 1: Nuclear transfer in Rana. How did the age of the donor nucleus affect the potency?

Answer 13

As age of donor nucleus increased, success rate decreased

Question 14

Testing nuclear potency 2: Nuclear transfer in Xenopus

Answer 14

Nucleus from gut cell of albino tadpole into pigmented egg

Only one half of embryo cleaved, so took nuclei from cleaved cells and put into e.g. –> all albino tadpoes

Question 15

Criticisms of nuclear transfer in Xenopus

Answer 15

• Low success rate
• Could be germ cells
• Gut epithelium not fully differentiated

Question 16

Testing nuclear potency 3: Nuclear transfer from adult skin cells in Xenopus

Answer 16

Nucleus from adult skin cells into pigmented egg
Check for gratin expression
Showed that the skin cells could be reprogrammed to become pluripotent

Question 17

Testing nuclear potency 4: Cloning sheep

Answer 17

Mammary epithelial cell (white sheep) and enucleated egg (black sheep) fused by electric current then culture to embryo and transfer to foster mum (black sheep).
Give birth to white sheep as nuclei came from white sheep

Question 18

Conclusions from testing nuclear potency

Answer 18

• Most cells lose potency during development
• Stem cells can remain multipotent
• Nuclei retain potency (under special circumstances)/ Genome constant

Question 19

Exceptions to nucelar potency conclusions

Answer 19

Parascaris - roundworm parasite

• Chromosome diminution
• Somatic cells lose genes
• Germplasm retains potency

Humans
- Immunoglobulin gene rearrangements in differentiated B lymphocytes

Question 20

In parascaris, what retains potency?

Answer 20

Germplasm

Question 21

In what cells does immunoglobulin gene rearrangements occur?

Answer 21

B lymphocytes

Question 22

Experiments that test nuclear potency

Answer 22

1) Nuclear transfer in Rana
2) Nuclear transfer in Xenopus
3) Nuclear transfer in Xenopus
4) Nuclear transfer in sheep

Question 23

What is used in nuclear transfer in Rana?

Answer 23

Enucleated egg cell and nuclei from cell of blast from embryo

Question 24

What is used in nuclear transfer in Xenopus?

Answer 24

• Nucleus from gut cell of albino tadpole and pigmented, enucleated egg
• Nucleus from adult skin cells and enucleated egg

Question 25

What is used in nuclear transfer in sheep?

Answer 25

Enucleated egg from black sheep and nucleus of mammary epithelial cells from white sheep