Stem cells and society Flashcards
What are the two categories of technologies affecting stem cells?
1) Genetic technologies
2) Reproductive technologies
What are 3 genetic technologies affeccting stem cells?
- Sequencing
- Genetic manipulation
- Artificial chromosomes1
What are 5 reproductive technologies affecting stem cells?
- IVF
- ES cells
- iPS cells
- In vitro gametogenesis
- Cloning
What is a transgenic organism?
- Organism whose genome has been altered by the transfer of a gene/ genes from another species or breed.
What are the 5 steps of generating a transgenic?
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
What is a chimera?
A single organism composed of cells with distinct genotypes
What are the features of pronuclear injection?
- Site of integration is random
- Number of copies integrated is random
(but multiple copies) - In a ferilised oocyte
- Generates a transgenic
What are the features of knockouts?
- Targeted gene insertion (specific integration
- Single copy
- Positive and negative selection
- In a blastocyst
- Generates a transgenic
What are HACs and what is their structure?
- 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
What are 3 features of HACs?
- Self-replication and self-segregating
- Behaves as a stable chromosome
- Independent from the chromosomes of the host cells
Why are ZFNs and TALENs difficult to design?
They rely on protein/DNA interaction
Why are CRISPR/CAS easy to design?
Use RNA to bind to DNA
What does CRISPR stand for?
Clustered regularly interspaced short palindromic repeats
What does CAS stand for?
- CRISPR-associated system
- It is a nuclease
In what 4 ways can CRISPR/CAS 9 edit the genome?
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
How can cas9 be manipulated to the advantage of scientists?
- 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
What are the 11 steps of in vitro fertilisation?
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
What is cloning described as?
Resetting the cell state by manipulating the nucleus
What 3 things must be reset during cloning and how?
1) Gene expression
- Somatic genes turned off
- Embryonic genes turned on
2) Methylation
- Reset back to ‘totipotent’ configuration
3) Chromatin
- Remodelled
How is pluripotency induced?
1) Viral transduction to differentiated somatic cells
2) Selection with drugs
3) Colony formation
What enzymes can cut DNA precisely?
TALENS
ZFN
CRISPR/ Cas9
