Stem cells and society

Question 1

What are the two categories of technologies affecting stem cells?

Answer 1

1) Genetic technologies

2) Reproductive technologies

Question 2

What are 3 genetic technologies affeccting stem cells?

Answer 2

• Sequencing
• Genetic manipulation
• Artificial chromosomes1

Question 3

What are 5 reproductive technologies affecting stem cells?

Answer 3

• IVF
• ES cells
• iPS cells
• In vitro gametogenesis
• Cloning

Question 4

What is a transgenic organism?

Answer 4

• Organism whose genome has been altered by the transfer of a gene/ genes from another species or breed.

Question 5

What are the 5 steps of generating a transgenic?

Answer 5

1) Transfect ES cells (infect with free nucleic acid)
2) Genotype ES cells and selecct the correctly targeted cells
3) ES cells microinjected into a host blastocyst
4) Blastocyst transfered into pseudo-pregnant (false pregnant)female
5) Chimeric mice produced and crossed for germline transmission

Question 6

What is a chimera?

Answer 6

A single organism composed of cells with distinct genotypes

Question 7

What are the features of pronuclear injection?

Answer 7

• Site of integration is random
• Number of copies integrated is random
  (but multiple copies)
• In a ferilised oocyte
• Generates a transgenic

Question 8

What are the features of knockouts?

Answer 8

• Targeted gene insertion (specific integration
• Single copy
• Positive and negative selection
• In a blastocyst
• Generates a transgenic

Question 9

What are HACs and what is their structure?

Answer 9

• Human artificial chromosomes
• Used to insert many genes into cells

Consisting of:
- Replication origin (ness. for DNA duplication)

• Centromere (to enable propper chromosome segregaiton)
• Telomere (protecting the ends of liner chromosomes

Question 10

What are 3 features of HACs?

Answer 10

• Self-replication and self-segregating
• Behaves as a stable chromosome
• Independent from the chromosomes of the host cells

Question 11

Why are ZFNs and TALENs difficult to design?

Answer 11

They rely on protein/DNA interaction

Question 12

Why are CRISPR/CAS easy to design?

Answer 12

Use RNA to bind to DNA

Question 13

What does CRISPR stand for?

Answer 13

Clustered regularly interspaced short palindromic repeats

Question 14

What does CAS stand for?

Answer 14

• CRISPR-associated system

- It is a nuclease

Question 15

In what 4 ways can CRISPR/CAS 9 edit the genome?

Answer 15

1) Gene knockouts
- Insert mutations or excise sequences

2) Gene repression/ activation
- Control a gene

3) Gene alteration
- Create a new version of a gene

4) Gene insertion
- Insert new DNA into the genome

Question 16

How can cas9 be manipulated to the advantage of scientists?

Answer 16

• Can remove nuclease activity
• Cas9 still is guided to the target by target RNA
• Can put additional features onto the Cas9
• Eg. RNA modifying enzymes, repressors, activators
• Target RNA can be synthetically made in order to make the cas9 target specific genes

Question 17

What are the 11 steps of in vitro fertilisation?

Answer 17

1) Synchronise and stimulate follicular development
2) Stimulate pre-ovulatory follicular maturation
3) Retrieve cumulus/ oocytes
4) Find ova, wash, place under oil
5) Transfer ova to sperm solution and incubate
6) Capacitate sperm
7) Perform ISIC if needed
8) Check zygote for fetilisation
9) Inccubate to 4-cell or blastocyst stage
10) Perform PGD if needed
11) Transer 1-2 embryos to uterine

Question 18

What is cloning described as?

Answer 18

Resetting the cell state by manipulating the nucleus

Question 19

What 3 things must be reset during cloning and how?

Answer 19

1) Gene expression
- Somatic genes turned off
- Embryonic genes turned on

2) Methylation
- Reset back to ‘totipotent’ configuration

3) Chromatin
- Remodelled

Question 20

How is pluripotency induced?

Answer 20

1) Viral transduction to differentiated somatic cells
2) Selection with drugs
3) Colony formation

Question 21

What enzymes can cut DNA precisely?

Answer 21

TALENS
ZFN
CRISPR/ Cas9