Stem cells & gene splicing

Question 1

Gene splicing & 3 approaches - 4

Answer 1

1. Knocking out/ reducing the expression of a gene.
   Most approaches include:
2. Antisense(knockdown)
3. Short interference RNAs(knockdown)
4. CRISPR(knockout)

Question 2

Gene knockdown vs Gene knockout

Answer 2

Gene Knockout: Permanent change in DNA leading to complete loss of function of a gene, effectively erasing it from the organism’s genome.

Gene Knockdown: Temporary decrease in gene expression, without completely eliminating the gene.

Question 3

Gene splicing: Antisense - 5

Answer 3

1. Single stranded RNA/DNA complementary (antisense) to target mRNA.
2. Uses predictive RNA structure programmes to target areas of accessible single stranded mRNA
3. Difficult to identify effective antisense sequences & ergo short interference RNAs used when targeting mRNAs in cell models.
4. Used for non-coding RNAs such as miRNA & long non-coding RNAs that are less amenable to siRNA mediated RNA interference.
5. Often employed in animal models & as therapeutics due to pharmacologically stability

Question 4

RNAi - 4

Answer 4

1. RNA interference is highly conserved
2. Efficient RNAi seen in mammals following exposure to short dsRNA (short interference RNAs).
3. Target coding regions of the mRNA
4. Design one strand that is complementary (antisense) to the target mRNA

Question 5

First two phases of CRISPR - 7

Answer 5

Phase 1:
1. Viral DNA fragments inserted into bacterial genome.
2. After infection, viral cleaved by Cas complex to make short spacer regions that are inserted into the CRISPR region of the bacterial genome.
Phase 2
3. Detection & destruction of virus upon re-infection
4. CRISPR region is transcribed
5. tacrRNA binds to the primary transcript & cleaved to produce crRNA (cRNA recognises a specific virus)
6. crRNA guides Cas9 protein to complementary virus DNA.
7. Cas9 then cleaves viral DNA & destroys the virus.

Question 6

CRISPR used to cleave human DNA & produce gene knockdown - 6

Answer 6

1. Requires expression of Cas9 protein
2. Design gRNA (guide RNA, a single hairpin RNA sequence designed around PAMs) targets Cas9 to target gene region (replaces crRNA)
3. Cas9 binds to PAM (protospace adjacent motif of 3 nucleotides) regions on DNA binding (e.g. NGG)
4. Following DNA cleavage, cell attempts to repair w/ error prone Non-homologous end joining or Homology directed repair
5. Non-homologous end joining can lead to insertion of extra nucleotides.
6. Homology directed repair (HDR) allows addition of large DNA sequences (that you have transfected into your cells)

Question 7

Gene delivery - 8

Answer 7

Transfection:
1. Mix antisense, siRNA or gRNA with transfection reagent
2. Add transfection reagent to suspended cells
3. For Adherent cells - layer transfection reagent on bottom of plate then add cells (negative transfection)

Major issues:
4. toxicity (need optimise conditions)
5. poor transfection of primary cells

Other methods:
6. Electroporation to produce pores electrically - but can kill desired cells

Viral Entry:
7. Lentivirus - integrated into DNA providing stable transfection
8. Adenoviral expression - not integrated into DNA & transient transfection

Question 8

Advantages and disadvantages of different gene silencing techniques?

Answer 8

Adv:
1. Only require knowledge of the mRNA or DNA sequence
2. potent knockdown of mRNA (will not always result in changes at protein level)
3. Cheap & easy to identify effective antisense, siRNA & gRNA sequences
4. siRNA & CRISPR is amenable to high throughput library screening

Dis:
1. Delivery - oligonucleotides are large & -ve charged, so delivery system must be effective. Need vesicles with +ve lipids to stick to the plasma membrane or viral vectors e.g as adenovirus/lentivirus
2. May induce immune response
3. Maybe no corelation between mRNA & protein levels - how much protein knockdown is required to see phenotypic effect?, how stable is the protein?

Question 9

Totipotent stem cell

Answer 9

Totipotent: Can form all tissues required for an organism including extra-embryonic tissue

Question 10

Pluripotent stem cell

Answer 10

Pluripotent: Can form all cells comprising an adult organism, e.g. embryonic stem cells

Question 11

Multipotent stem cell

Answer 11

Multipotent: Can form a limited number of cell types, e.g. most adult stem cells

Question 12

Unipotent stem cell

Answer 12

Unipotent: Can only form one type of cell, e.g. committed progenitor cells

Question 13

Adult stem cell characteristics - 8

Answer 13

1. Can proliferate (self-renewal) but this will not continue indefinitely
2. Can only differentiate into a limited number of cells (multipotent or unipotent)
3. Located in organs & tissues in very small numbers in areas called ‘stem cell niches’
4. Important in organ & tissue homeostasis
5. Vast majority of body cells are terminally differentiated
6. Replace worn out or lost cells
7. Difficult to identify and culture
8. Demonstrate ability to re-populate organ or tissue following re-injection

Question 14

Some applications of adult stem cells - 5

Answer 14

1. Bone marrow transplants
2. Multiple sclerosis
3. Artificial Skin Grafts
4. Corneal Transplants
5. Age-Related Macular Degeneration (eye degradation)

Question 15

Characteristics of Embryonic stem cells - 3

Answer 15

1. Continue to proliferate almost indefinitely
2. Can be differentiated into all cell types (pluripotent)
3. Form tumours when injected into immuno-compromised mice

Question 16

Endoderm, Mesoderm & Ectoderm

Answer 16

Endoderm - form internal organs, pancreas, liver
Mesoderm - form blood, circulation, heart, muscle
Ectoderm - form nervous tissue, skin

Question 17

Cloning - 2

Answer 17

1. Remove nucleus from egg, inject in nucleus from somatic cell & expose to electrical shock
2. Need to produce cocktail of chemicals that drive the egg to divide past 16 cell stage (includes caffeine)

Question 18

Characteristics of inducted pluripotent stem cells (iPSC) - 5

Answer 18

1. No ethical issue since derived from adult cells
2. Can proliferate almost indefinitely
3. Can be differentiated into >200 types
4. Reduced chances of graft-versus-host since these are derived from individuals to be treated
5. May develop into cancers

Question 19

Transgenic animals - 2

Answer 19

1. A transgenic animal is an organism that has been genetically modified by the introduction of foreign DNA into its genome
2. Allows researchers to investigate disease states inaccessible in human patients, & allows development of new drugs/therapeutic approaches.

Question 20

Use of transgenic organisms - 5

Answer 20

1. To test gene function - typically gain of function e.g. action of oncogenes > tumour formation
2. To develop animal models of human disease e.g. role of cytokines in inflammatory conditions, IL-13
3. To tag (often fluorescent) & track genes of interest
4. For large scale production of therapeutic proteins e.g. antibodies in bovine milk
5. Agricultural purposes e.g. pesticide resistance

Question 21

How are transgenic organisms made - 2

Answer 21

1. Take a gene you want to express from the inner cell mass & stick on a promotor on the front of the gene.
2. Insert it onto an embryonic stem cell.

Question 22

Advantages of embryonic stem cells - 5

Answer 22

1. Can be grown for many generations
2. Transgenes can be inserted into a specific site
3. Presence of transgene can be detected using qRT-PCR
4. Transgenic embryonic stem cells can be rapidly produced & stored
5. Can use premade stem cells

Question 23

Tissue specific promoter

Answer 23

Include promoter that is driven by a transcription factor that is only expressed in specific cell/tissue.

Question 24

Inducible promoter

Answer 24

Cis-acting elements that allow timing, location & level of expression of a transgene to be expressed.