JD - Gene Transfer and Silencing

Question 1

What viruses are the preferred vehicle for gene transfer?

Answer 1

1. Retroviruses (RNA)
2. Adenoviruses (DNA)
3. Lentiviruses (RNA)
4. Adeno associated virus (DNA)

Question 2

What is the genetic material surrounded by in a virus?

Answer 2

Protein coat called a capsid

Question 3

How big is the retrovirus genome in size?

Answer 3

ssRNA, 7-9 kb in size

Question 4

What structures are present in the retrovirus genome? (5)

Answer 4

LTR- Long terminal repeat
Ψ- Packaging signal
GAG- Capsid proteins
POL- Reverse Transcriptase
ENV- Envelope protein

Question 5

What occurs to the retrovirus genome upon entry into cells? (2)

Answer 5

1) The virus loses its envelope and releases RNA

• Transcriptase also released

2) ssRNA ==> DNA

• Becomes integrated into the host chromosome

Question 6

How are viral vectors packaged?

Answer 6

Using a packaging cell line

Question 7

What are the main steps involved in PCL? (4)

Answer 7

1. Structural and viral proteins are transcribed and translated from the integrated retrovirus
2. Essential proteins are made for packaging e.g Capsid
3. The modified cell line is transfected with plasmids containing the viral genes of interest
4. Once the viral genome is packaged, the viral particles are released from the packaging cell line via budding

Question 8

What type of disorders can retroviral vectors be used to treat?

Answer 8

Immune e.g ADA

Question 9

What are some advantages and disadvantages of using retroviral vectors?

Answer 9

Advantages

• High efficiency of gene transfer
• High levels of expression

Disadvantages

• Max insert size 7-7.5kb
• Only infect dividing cells
• Problems with integration (toxic)

Question 10

What is an advantage of using lentiviruses rather than standard retroviruses? (2)

Answer 10

1. Can infect both dividing and non-dividing cells
2. Increased safety compared to earlier retroviruses

• Engineered to remove the strong promoters and enhancers in the LTRs

Question 11

What does RNAi help with?

Answer 11

Selectively inhibiting the mutant gene from being expressed

Question 12

What are the main 3 components of the gene silencing pathway?

Answer 12

1. Dicer (dsRNA ==> siRNA)
2. siRNA- short interfering RNA
3. RISC- RNA-induced silencing complex

Question 13

What are the main steps involved in RNAi silencing? (5)

Answer 13

1. dsRNA ==> siRNA (Dicer)
2. siRNA integrated into RISC
3. siRNA within RISC binds to mRNA sequence
4. Induces the cleavage and degradation of the target mRNA
5. Translation does not occur (silenced)

Question 14

What are some disadvantages of adenoviral vectors? (4)

Answer 14

•Adenoviral vectors do not insert the genes into the host chromosome

•Limited to only temporary protein expression

•Does not work well on dividing cells

•Requires repeat treatments