Lecture 15; Reproductive cloning

Question 1

What are the types of cloning?

Answer 1

Reproductive cloning
Therapeutic cloning
Molecular cloning

Question 2

Define reproductive cloning;

Answer 2

Creating a new individual for the purpose of reproduction (genetically identical from parents cell)

Question 3

Define therapeutic cloning;

Answer 3

Uses similar tehcnology to reproductive technology but with the purpose other than to create a new individual

Question 4

Define molecular cloning;

Answer 4

Usually refers to creating bacteria or other cells that contain an identical gene

Question 5

Give a brief overview of embryonic development;

Answer 5

The fertilised oocyte undergoes symmetric divisions in which each blastomere is essentially identical until the morula/blastocyst stage

Question 6

What are cloning technologies?

Answer 6

Embryo splitting
Somatic Cell Nuclear Transfer (SCNT)
Handmade cloning

Question 7

Describe cloning by embryo splitting;

Answer 7

• 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

Question 8

Describe what SCNT is

Answer 8

Fusing the nucleus of a somatic cell i.e epithelial cell or fibroblast with an unucleated oocyte (Cytoplast)

Question 9

Describe SCNT methodology

Answer 9

Oocyte, enucleated -> Cytoblast

Donor somatic cell or its nuclie are fused.

Electrically shocked to stimulate devision and thus growth

Question 10

Describe the first instance of SCNT;

Answer 10

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)

Question 11

Describe how dolly was produced;

Answer 11

Fusion of the nucleus from a mammary gland cell from a 6 year old sheep with an enucleated (cytoplast)

Question 12

Was SCNT effective?

Answer 12

Not particularly dolly was 1/277 reconstructs

Question 13

Where can cells for SCNT be obtained from?

Answer 13

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.

Question 14

When are best results of SCNT achieved?

Answer 14

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

Question 15

Describe the cloning phenotype of SCNT cows

Answer 15

Offspring produced by SCNT tended to have a consistant phenotype characterised by;

• Fetal overgrowth
• Musculoskeletal abnormalities
• Early death

Question 16

Describe the success of AgResearch with SCNT

Answer 16

133/998 transfered embryos resulted in delivery at term (13%)

67% of the 133 survived 3 months

Question 17

What did Japanese research suggest the overgrowth syndrome was caused by;

Answer 17

Overgrowth syndrome = higher birth weight = earlier death

Inappropriate expression of genes such as IGF i.e incorrect nuclear reprogramming

Question 18

Why would incorrect nuclear reprogramming occur?

Answer 18

• 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

Question 19

Describe DNA methylation

Answer 19

• 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

Question 20

Whats a real problem with cloning?

Answer 20

Ethical issues

Especially if many required culling off

Question 21

What happened to dolly?

Answer 21

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)

Question 22

Even though dolly died young, what happened to her offspring?

Answer 22

• Offspring had normal telomere length

Question 23

Why did dolly die?

Answer 23

• Dolly was physically fine for her age

- At 6.5 years she died from progressive lung disease and arthritis

Question 24

What may telomere length in clones depend on?

Answer 24

Telomere length in clones may depend on the tissue the nucleus is derived from - muscle versus oviduct or mammary gland.

Question 25

Does the ‘cloning phenotype’ extend to their reproduction?

Answer 25

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

Question 26

What is one difference of a reproducing clone?

Answer 26

• Milk production in clones is similar to that of karyoplast donor

Question 27

Whats an alternative to traditional SCNT?

Answer 27

Handmade cloning, does not require traditional expensive equipment

HMC can be high throughput

Question 28

Whats the problems with HMC?

Answer 28

• 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)

Question 29

What is HMC all about?

Answer 29

Preparations of cytoplasts / oocyte enucleation

Question 30

Describe HMC process of enculeation;

Answer 30

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

Question 31

What makes HMC hand made?

Answer 31

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

Question 32

What can be done in HMC to ensure reasonable cytoplasm?

Answer 32

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

Question 33

What is done in HMC once the cells are prepared?

Answer 33

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.

Question 34

Why do we want to clone?

Answer 34

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

Question 35

Should cloning be performed on humans?

Answer 35

The ethics committee of the american society for reproductive medicine says NO

• Safety
• Unknown affects of SCNT

Question 36

Are cloned animals genetically modified?

Answer 36

No

• Mitochondria is maternal
• However they could be made trasngenic (GM)

Question 37

Why would we use transgenes in cloning?

Answer 37

Would use transgenes for;

• Medical Models
• Protein production i.e human lactoferrin
• Improved production animals-cloning lysozyme driven by beta casein.