Cloning and Genomic Equivalence Flashcards
What happens as a cell starts do differentiate?
Gradual restriction in potency
Why is there a restriction in potency?
- Gene loss
- Differential gene activity (largely this is the case)
What experiment provides evidence for differential gene activity in potency?
Lens regneration in the newt
Process of lens regeneration in the newt experiment
- Remove lens
- Proliferation of cells in dorsal iris
- Synthesis of crystallin proteins (found in lens)
Iris cells nuclei have genes for crystallin but wouldn’t normally express it
What proteins are expressed in the iris to regenerate the lens in newts?
Crystallin proteins
What is a heterokaryon?
A heterokaryon is a multinucleate cell that contains genetically different nuclei
Human liver cell transplanted into rat muscle cell
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
What did results of a human liver cell transplanted into a rat muscle cell show?
Liver gene silenced and human muscle gene switched on. Gene wasn’t lost but cytoplasm of muscle cell induced change
Nucleus from Xenopus kidney cells into Pleurodeles oocyte (newt)
Kidney-specfific genes were silenced. Expressed oocyte-specific genes
What does the fact that differentiated Xenopus kidney cells expressed oocyte-specific genes when transferred?
Genetic info is not lost after cells differentiate
Conclusions about gene expression
- 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
Testing nuclear potency 1: Nuclear transfer in Rana (frog)
Enucleated egg cell and nucleus from cell in blastula stage of an embryo
Shows cells lose potency but nuclei retain it
Testing nuclear potency 1: Nuclear transfer in Rana. How did the age of the donor nucleus affect the potency?
As age of donor nucleus increased, success rate decreased
Testing nuclear potency 2: Nuclear transfer in Xenopus
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
Criticisms of nuclear transfer in Xenopus
- Low success rate
- Could be germ cells
- Gut epithelium not fully differentiated
Testing nuclear potency 3: Nuclear transfer from adult skin cells in Xenopus
Nucleus from adult skin cells into pigmented egg
Check for gratin expression
Showed that the skin cells could be reprogrammed to become pluripotent
Testing nuclear potency 4: Cloning sheep
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
Conclusions from testing nuclear potency
- Most cells lose potency during development
- Stem cells can remain multipotent
- Nuclei retain potency (under special circumstances)/ Genome constant
Exceptions to nucelar potency conclusions
Parascaris - roundworm parasite
- Chromosome diminution
- Somatic cells lose genes
- Germplasm retains potency
Humans
- Immunoglobulin gene rearrangements in differentiated B lymphocytes
In parascaris, what retains potency?
Germplasm
In what cells does immunoglobulin gene rearrangements occur?
B lymphocytes
Experiments that test nuclear potency
1) Nuclear transfer in Rana
2) Nuclear transfer in Xenopus
3) Nuclear transfer in Xenopus
4) Nuclear transfer in sheep
What is used in nuclear transfer in Rana?
Enucleated egg cell and nuclei from cell of blast from embryo
What is used in nuclear transfer in Xenopus?
- Nucleus from gut cell of albino tadpole and pigmented, enucleated egg
- Nucleus from adult skin cells and enucleated egg
What is used in nuclear transfer in sheep?
Enucleated egg from black sheep and nucleus of mammary epithelial cells from white sheep
