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Recombinant Viral Techniques – Detailed Notes


Recombinant Viral Techniques – Detailed Notes

1. Definition

Recombinant viral techniques involve using viruses as vectors to deliver foreign genes into host cells.
The foreign gene is engineered into the viral genome, allowing the virus to infect target cells and express the gene.
Widely used in gene therapy, functional genomics, vaccine development, and protein production.

2. Principle

A viral genome is modified in vitro to carry a gene of interest.
Viral genes responsible for replication or pathogenicity may be deleted or inactivated to ensure safety.
The recombinant virus infects target cells, delivering the foreign gene.
The gene may be expressed transiently or stably, depending on the virus type.
Key Concept:
Viruses act as natural delivery vehicles that efficiently enter cells, bypassing the limitations of physical or chemical gene delivery methods.
3. Types of Recombinant Viral Vectors

Viral vectors can be classified based on genome type and integration behavior.
A. Retroviral Vectors
Genome: RNA
Mechanism: Reverse-transcribed into DNA → integrated into host genome
Expression: Stable (long-term)
Advantages: Stable integration, useful for dividing cells
Limitations: Cannot infect non-dividing cells efficiently; risk of insertional mutagenesis
Examples:
Moloney Murine Leukemia Virus (MoMLV) vectors
Lentiviral vectors (HIV-based) – can infect dividing & non-dividing cells.


B. Adenoviral Vectors


Genome: Double-stranded DNA
Mechanism: Remains episomal (does not integrate)
Expression: High-level transient expression
Advantages: High efficiency, infects dividing and non-dividing cells
Limitations: Transient expression; immunogenicity

Examples:

Human adenovirus serotype 5 (Ad5) vectors
C. Adeno-Associated Viral (AAV) Vectors
Genome: Single-stranded DNA
Mechanism: Mostly episomal; sometimes integrates at specific site
Expression: Long-term, low immunogenicity
Advantages: Safe, stable expression, minimal immune response
Limitations: Limited DNA cargo (~4.5 kb)


D. Herpes Simplex Virus (HSV) Vectors


Genome: Large double-stranded DNA
Mechanism: Episomal in neurons, non-integrating
Advantages: Can carry large genes (~30–40 kb)
Limitations: Complex engineering, potential cytotoxicity
E. Other Viral Vectors
Vaccinia virus – used in vaccines and gene expression
Retrovirus-derived vectors – gene therapy and functional genomics
4. Steps in Recombinant Viral Vector

Construction
Selection of virus type based on host cell and application
Isolation and modification of viral genome
Delete pathogenic genes
Insert foreign gene under suitable promoter
Packaging of recombinant virus
In packaging cell lines (produce viral particles without replication-competent virus)
Infection of target cells
Expression and analysis of foreign gene
Reporter genes (GFP, LacZ) often used initially
5. Promoters Used in Recombinant Viral Vectors


Strong viral promoters ensure high-level expression:
CMV promoter (Cytomegalovirus) – adenoviral and AAV vectors
RSV promoter (Rous Sarcoma Virus) – retroviral vectors
35S promoter (for plant viral vectors, e.g., CaMV)
6. Applications of Recombinant Viral Techniques


6.1 Gene Therapy
Correct defective genes in patients with genetic disorders.
Examples:
SCID (Severe Combined Immunodeficiency) – retroviral vectors
Hemophilia – AAV vectors


6.2 Vaccine Development


Recombinant viral vectors deliver antigen genes to induce immunity.
Examples:
Recombinant adenovirus expressing SARS-CoV-2 spike protein
Vaccinia virus-based vaccines

6.3 Functional Genomics

Study gene function by overexpression, silencing, or mutation.
Example: Lentiviral vectors for shRNA delivery

6.4 Protein Production

High-level expression of therapeutic proteins in cultured cells.
Example: Recombinant adenovirus expressing insulin or growth factors

6.5 Cancer Therapy


Oncolytic viruses engineered to target and kill tumor cells while delivering therapeutic genes.

7. Advantages of Recombinant Viral Vectors


Efficient gene delivery into target cells
High-level expression (adenovirus, HSV)
Stable integration (retrovirus, lentivirus)
Can target dividing and non-dividing cells
Useful for both research and therapeutic applications
8. Limitations and Safety Concerns


Potential immune response (especially adenovirus)
Risk of insertional mutagenesis (retroviral vectors)
Limited cargo capacity (AAV ~4.5 kb)
Complex production and handling for large viruses (HSV, vaccinia)
Biosafety considerations for human application.


Recombinant Viral Techniques – 50 MCQs


Recombinant viral techniques involve:
a) Using viruses to delete host genes
b) Using viruses to deliver foreign genes ✅
c) Using bacteria to deliver proteins
d) Using chemicals for gene editing


The foreign gene in recombinant viral techniques is inserted into:

a) Host DNA only
b) Viral genome ✅
c) RNA only
d) Plasmid only

Retroviral vectors have:
a) DNA genome
b) RNA genome ✅
c) Protein genome
d) Single-stranded DNA

Adenoviral vectors typically provide:
a) Stable integration
b) Transient high-level expression ✅
c) Low expression
d) No expression


AAV vectors are preferred because:
a) High immunogenicity
b) Low immunogenicity ✅
c) Very large cargo capacity
d) Only infect bacteria
Lentiviral vectors are a type of:
a) Retrovirus ✅
b) Adenovirus
c) Herpesvirus
d) AAV
Recombinant viral vectors are used in:
a) Gene therapy ✅
b) Vaccine development ✅
c) Functional genomics ✅
d) All of the above ✅
Retroviral vectors integrate into host genome via:
a) Random integration ✅
b) Episomal replication
c) RNA interference
d) Non-specific protein binding
Adenoviral vectors remain:
a) Integrated
b) Episomal ✅
c) Mutated
d) Silent
AAV vectors can carry foreign DNA of up to:
a) 2 kb
b) 4–4.5 kb ✅
c) 10 kb
d) 50 kb
HSV vectors are useful for delivering:
a) Small genes only
b) Large genes (30–40 kb) ✅
c) Only RNA
d) Only plasmids
Which promoter is commonly used in recombinant viral vectors?
a) CMV promoter ✅
b) T7 promoter
c) Lac promoter
d) CaMV 35S promoter
Recombinant viral vectors are efficient because:
a) Viruses naturally infect cells ✅
b) DNA degrades quickly
c) Only bacterial cells are infected
d) None of the above
Retroviral vectors cannot efficiently infect:
a) Dividing cells
b) Non-dividing cells ✅
c) Mammalian cells
d) Stem cells
Adenoviral vectors are highly immunogenic, which:
a) Helps expression
b) Limits repeated use ✅
c) Increases cargo capacity
d) Prevents infection
Lentiviral vectors can infect:
a) Only dividing cells
b) Both dividing and non-dividing cells ✅
c) Only neurons
d) Only bacteria
Applications of recombinant viral vectors include:
a) Protein production ✅
b) Cancer therapy ✅
c) Gene therapy ✅
d) All of the above ✅
Oncolytic viruses are:
a) Viruses engineered to infect bacteria
b) Viruses that target and kill cancer cells ✅
c) Non-replicating viral vectors
d) Only used in plants
Advantages of recombinant viral vectors include:
a) Efficient delivery ✅
b) High-level expression ✅
c) Stable or transient expression ✅
d) All of the above ✅
Limitations include:
a) Immune response ✅
b) Insertional mutagenesis (retrovirus) ✅
c) Limited cargo size (AAV) ✅
d) All of the above ✅
Which viral vector is safest for human gene therapy?
a) Adenovirus
b) AAV ✅
c) Retrovirus
d) HSV
Adenovirus vectors can infect:
a) Only dividing cells
b) Both dividing and non-dividing cells ✅
c) Only stem cells
d) Only bacteria
Retroviral vectors are most suitable for:
a) Short-term expression
b) Stable long-term expression ✅
c) Only transient assays
d) Plant cells only
HSV vectors are particularly useful in:
a) Neurons ✅
b) Liver cells only
c) Bacteria
d) Plant leaves
AAV vectors integrate at:
a) Random sites
b) Specific site on chromosome 19 ✅
c) Mitochondrial DNA
d) RNA genome
CMV promoter in viral vectors ensures:
a) Low expression
b) Strong constitutive expression ✅
c) Only tissue-specific expression
d) No expression
Recombinant adenovirus vectors are commonly used for:
a) Human gene therapy ✅
b) Plant transformation
c) Bacterial expression
d) RNA virus delivery
Packaging cell lines are used to:
a) Replicate viral genome ✅
b) Express selectable markers
c) Transform bacteria
d) Destroy viral vectors
Herpesvirus vectors can carry:
a) ~4 kb DNA
b) 30–40 kb DNA ✅
c) 50–100 bp DNA
d) Only RNA
Lentiviral vectors are derived from:
a) HIV ✅
b) MoMLV
c) Adenovirus
d) HSV
Recombinant viral vectors can be used in:
a) Human therapy ✅
b) Functional genomics ✅
c) Vaccine development ✅
d) All of the above ✅
Insertional mutagenesis risk is highest in:
a) Adenovirus
b) Retroviral vectors ✅
c) AAV
d) HSV
Transient expression is typical for:
a) Retrovirus
b) Adenovirus ✅
c) Lentivirus
d) None
Recombinant viral vectors are engineered to:
a) Enhance viral pathogenicity
b) Delete pathogenic genes ✅
c) Increase viral virulence
d) Prevent infection
AAV vectors have low immunogenicity because:
a) They replicate fast
b) They are non-pathogenic ✅
c) They integrate randomly
d) They express toxic proteins
Recombinant viral vectors can be used to deliver:
a) Coding genes ✅
b) shRNA for silencing ✅
c) Reporter genes ✅
d) All of the above ✅
Recombinant adenovirus expression is usually:
a) Long-term
b) Short-term ✅
c) Only in bacteria
d) Only in plants
HSV vectors are particularly useful for:
a) Large gene delivery ✅
b) Bacterial expression
c) Plant protoplasts
d) Short peptides only
Recombinant viral vectors in vaccine development work by:
a) Killing bacteria
b) Delivering antigen genes ✅
c) Destroying RNA
d) Integrating into patient chromosomes only
A major advantage of lentiviral vectors is:
a) Transient expression
b) Infection of non-dividing cells ✅
c) High immunogenicity
d) Very small DNA cargo
Recombinant viral vectors are preferred over plasmids because:
a) Lower efficiency
b) Natural infection allows efficient delivery ✅
c) Limited applications
d) Only work in plants
Viral vectors can be designed to carry:
a) Single gene
b) Multiple genes ✅
c) shRNA sequences
d) All of the above ✅
Which viral vector is used in SCID gene therapy?
a) Adenovirus
b) Retrovirus ✅
c) AAV
d) HSV
Oncolytic viruses can:
a) Only deliver genes
b) Deliver genes and selectively kill tumor cells ✅
c) Infect bacteria
d) Only infect neurons
A major limitation of adenovirus vectors is:
a) Low expression
b) High immunogenicity ✅
c) Small DNA cargo
d) Cannot infect human cells
Recombinant viral vectors require:
a) DNA integration always
b) Engineering in vitro ✅
c) Random infection
d) No safety precautions
Reporter genes commonly used in viral vectors include:
a) GFP ✅
b) LacZ ✅
c) Luciferase ✅
d) All of the above ✅
Stable gene expression is typical for:
a) Retrovirus ✅
b) Adenovirus
c) HSV
d) None
Recombinant viral vectors are used in functional genomics to:
a) Overexpress genes ✅
b) Silence genes ✅
c) Study promoter activity ✅
d) All of the above ✅
Overall advantage of recombinant viral techniques:
a) Random mutation only
b) Efficient, targeted gene delivery with stable or transient expression ✅
c) Only bacteria transformation
d) Only plant transformation





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