Viral Vector Essay Exercise

Instructions:

Use the following essay template to write an essay no greater than 2000 words.

Complete the following checklist for each point in the template

• Statement: Re-write the template point into your own words, be sure to use professional scientific language. This needs a reference, to show that it is a respected, peer-reviewed opinion.
  
• Explanation: Explain the concept to make it easier to understand, be concise. This does not need referencing and should reflect your understanding of the statement.
  
• Example: Provide an example, include a reference. This may be a particular gene, experiment completed, journal published. Needed to validify your statment.
  

Format the essay, proof read it and print it out to add to supplement your learning.

Alternatively this could be an assessment or classroom task.

Each numbered list begins a new paragraph:

Viral Vectors

Introduction:

• Viral vectors may be used to study gene function
• Over express target genes
• Generate animal models
• RNA interference, gene knockout administered at any developmental period not host-specific
• Spatially selective
• High levels of transgene
• Can combine with transgenic models for contraindications/synergism
• Rescue phenotype to confirm

1) RNAi

Post transcriptional gene silencing using dsRNA to knock down gene expression used for gene function tests.
Achieved by direct delivery of:

• siRNA
• shRNA
• miRNA
  

Mechanism:

1. dsRNA introduced to cell
2. DICER cleaves into ~20bp siRNA’s
3. siRNA’s introduced to into RISC
4. siRNA’s undergo strand separation
5. Hybridization with complementary mRNA
6. Argonuate cleaves mRNA within RISC

2) siRNA’s

• 21-23bp dsRNA with 3’ dinucleotide overhang
• Algorithms used to design siRNA’s
• Several coupled to cover gene of interest
• Delivery: transfection, electroporation, injection
• Transient knockdown (3-7 days)

3) shRNA

• Hairpin siRNA
• Sense and antisense strand of siRNA
• Promoter/enhancer Pol. III, Pol. II
• Termination Pol. III: string of T’s, minimal SV40 poly A signal
• Delivery by plasmids, vectors
• Long term expression

4) miRNA

• Endogenous 20bp RNA’s from introns
• Regulates expression by binding to 3’ untranslated region on exons
• Regulate cell growth, proliferation, death, developments miRBase: contains >3000 miRNA regulating >30% of exons
• Mechanism:
1. Transcription of pri-mRNA
2. Drosha processes into pre-mRNA
3. Export pre-mRNA into cytoplasm
4. DICER processing
5. Strand selection by RISC
• Imperfect duplex = Repression (mRNA level intact)
• Perfect duplex = mRNA degeneration
• Use Invitrogen “Block-it” to design RNAi’s for gene function studies

Conclusion

Potentially new therapeutic strategy

Marking schedule:

Paragraph	Marks	Total Marks	Teachers Notes
Introduction		/10
Para 1:		/20
Para 2:		/20
Para 3:		/20
Para 4:		/20
Conlusion:		/5
Presenta

100marks. 1% per mark.

• A: 80+
• B:65+
• C:50+