Lecture 15; Reproductive cloning Flashcards
What are the types of cloning?
Reproductive cloning
Therapeutic cloning
Molecular cloning
Define reproductive cloning;
Creating a new individual for the purpose of reproduction (genetically identical from parents cell)
Define therapeutic cloning;
Uses similar tehcnology to reproductive technology but with the purpose other than to create a new individual
Define molecular cloning;
Usually refers to creating bacteria or other cells that contain an identical gene
Give a brief overview of embryonic development;
The fertilised oocyte undergoes symmetric divisions in which each blastomere is essentially identical until the morula/blastocyst stage
What are cloning technologies?
Embryo splitting
Somatic Cell Nuclear Transfer (SCNT)
Handmade cloning
Describe cloning by embryo splitting;
- Identical twins result from embryo splitting
- Mammalian embryo splitting works but only to a limited number of viable embryos can be produced from the starting embryo
- This is b/c only those blastomeres prior to morula stage are totipotent and able to form all cell lineages of an individual
Used for agricultural purposes
Describe what SCNT is
Fusing the nucleus of a somatic cell i.e epithelial cell or fibroblast with an unucleated oocyte (Cytoplast)
Describe SCNT methodology
Oocyte, enucleated -> Cytoblast
Donor somatic cell or its nuclie are fused.
Electrically shocked to stimulate devision and thus growth
Describe the first instance of SCNT;
Fusion of a whole cell from an 8 cell embryo with enucleated oocytes and produced live offspring.
Donor cell was embryonic not somatic at this point (1986)
Describe how dolly was produced;
Fusion of the nucleus from a mammary gland cell from a 6 year old sheep with an enucleated (cytoplast)
Was SCNT effective?
Not particularly dolly was 1/277 reconstructs
Where can cells for SCNT be obtained from?
Somatic cells can be obtained from slaughtered animals and then can be matured in vivo
Alternatively oocytes can be obtained from living animals with ultrasound guidance.
When are best results of SCNT achieved?
Best results with SCNT are achieved when the donor nucleus is in either G0 or G1
- This can be achieved by culturing the donor cells in the absence of serum, by contact inhibition or other methods
Describe the cloning phenotype of SCNT cows
Offspring produced by SCNT tended to have a consistant phenotype characterised by;
- Fetal overgrowth
- Musculoskeletal abnormalities
- Early death
Describe the success of AgResearch with SCNT
133/998 transfered embryos resulted in delivery at term (13%)
67% of the 133 survived 3 months
What did Japanese research suggest the overgrowth syndrome was caused by;
Overgrowth syndrome = higher birth weight = earlier death
Inappropriate expression of genes such as IGF i.e incorrect nuclear reprogramming
Why would incorrect nuclear reprogramming occur?
- Sperm DNA is hypermethalated (epigenetics)
- Oocyte DNA is hypomethalated
- During normal embryogenesis DNA marks are removed and reprogrammed
The donor Karoyplast (nucleus) in SCNT has different epigenetic characteristics to that of gametes and must be reprogrammed to embryonic form
Describe DNA methylation
- Methyl group is added to the DNA backbone at C usually in a sequence of clusters of CpG sequences.
- CpG islands are often located in the proximity of promoters and they turn transcription off
- Genes important to embryogenesis retain some methylation
Whats a real problem with cloning?
Ethical issues
Especially if many required culling off
What happened to dolly?
She died young
At one year of age her telomere length was similar to that of the six year old mammory tissue she was cloned from (shortened)
Even though dolly died young, what happened to her offspring?
- Offspring had normal telomere length
Why did dolly die?
- Dolly was physically fine for her age
- At 6.5 years she died from progressive lung disease and arthritis
What may telomere length in clones depend on?
Telomere length in clones may depend on the tissue the nucleus is derived from - muscle versus oviduct or mammary gland.
Does the ‘cloning phenotype’ extend to their reproduction?
Despite the abormalities that appear in many cloned animals, those clones that reach reproductive age, reproduce normally.
- Similar fertility rates to normal
- Cloning phenotype not in offspring
- Epigentic defects must correct during gamatogensis in clones
What is one difference of a reproducing clone?
- Milk production in clones is similar to that of karyoplast donor
Whats an alternative to traditional SCNT?
Handmade cloning, does not require traditional expensive equipment
HMC can be high throughput
Whats the problems with HMC?
- Done with Zona free oocytes there may be problems associated with development of the embryo without the protective environment of the zona.
- Smaller number of cells in ICM
- Exposure to toxic substances etc (lac of zona protection)
What is HMC all about?
Preparations of cytoplasts / oocyte enucleation
Describe HMC process of enculeation;
Chemical enucleation; Blocking topoisomerase 2 during metaphase 1 prevents separation of the chromosomes and all DNA is shed from the oocyte in the polar body.
- Other chemicals can also be used to aggregate the chromosomes so the expelled or can be removed easily
What makes HMC hand made?
IN a zona free oocyte the chromosomes protrude in a bulge in the oocyte. Using a sharp blade the oocyte can be cut by hand with a visualised using a stereomicroscope - hence handmade
What can be done in HMC to ensure reasonable cytoplasm?
Because cutting the oocyte by hand results in a smaller cytoplasm or hemi-cytoplasm somtimes two hemi-cytoplasts are fused to give a reasonable volume of cytoplasm
What is done in HMC once the cells are prepared?
The hemi-cytoplasts are briefly added to PHA which induces stickiness then electrofused with either a donor cell or nucelus
The reconstruct is then chemically activated.
Why do we want to clone?
1) High value genetics i.e high grade beef
2) Understand basic science of cloning
3) Conservation
4) Valuable medical models
5) Reproduction for infertile couples/individuals
Should cloning be performed on humans?
The ethics committee of the american society for reproductive medicine says NO
- Safety
- Unknown affects of SCNT
Are cloned animals genetically modified?
No
- Mitochondria is maternal
- However they could be made trasngenic (GM)
Why would we use transgenes in cloning?
Would use transgenes for;
- Medical Models
- Protein production i.e human lactoferrin
- Improved production animals-cloning lysozyme driven by beta casein.
