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Gene Transfer Technologies – Detailed Notes


Gene Transfer Technologies – Detailed Notes

1. Definition

Gene transfer is the process of introducing foreign DNA or genes into the genome of a target organism or cell.
It allows the expression of new traits, study of gene function, and production of therapeutic proteins.
Also known as gene delivery or genetic transformation.
2. Principles of Gene Transfer


Involves delivery of DNA or RNA into cells or organisms.
DNA can be integrated into the host genome or remain episomal (non-integrated).
The goal is stable or transient expression of the transferred gene.

Key considerations:

Vector – vehicle for carrying the gene
Target cell – plant, animal, microbial, or human cells
Delivery method – physical, chemical, or biological


3. Types of Gene Transfer

Gene transfer can be broadly classified into:
A. Natural Gene Transfer
Occurs in nature between organisms:
Transformation: Uptake of naked DNA by bacteria.
Transduction: DNA transfer via viruses (bacteriophages).
Conjugation: Transfer of plasmids via direct cell-to-cell contact.
B. Artificial/Engineered Gene Transfer
Done in laboratories to introduce desired genes:
Direct DNA transfer (Physical methods)
Vector-mediated transfer (Biological methods)
Chemical-mediated transfer

4. Methods of Gene Transfer
A. Physical Methods
Microinjection:
DNA is directly injected into the nucleus or cytoplasm of a cell using a fine glass micropipette.
Used in: animals (e.g., mice), plants (protoplasts)

Advantage: High precision; disadvantage: Labor-intensive, low throughput
Electroporation:
Brief electric pulses create temporary pores in the cell membrane, allowing DNA to enter.
Used in: bacteria, yeast, plant protoplasts, mammalian cells
Advantage: Efficient; limitation: Can cause cell death
Particle Bombardment (Biolistics / Gene Gun):
DNA-coated gold or tungsten particles are shot into target cells using high-pressure helium.
Widely used in plant genetic engineering.
Liposome-Mediated Transfer (Lipofection):
DNA is encapsulated in lipid vesicles (liposomes) that fuse with cell membranes.
Used in mammalian cells and some plant cells.
B. Biological Methods

Agrobacterium-Mediated Transformation (plants):
Agrobacterium tumefaciens transfers a portion of its Ti plasmid (T-DNA) into plant cells.
Widely used for stable genetic transformation of dicots.
Viral Vectors:
Modified viruses deliver genes into host cells.
Examples:
Retrovirus – integrates into host genome
Adenovirus – transient expression
Adeno-associated virus (AAV) – low immunogenicity
Applications: gene therapy, functional genomics
Bacterial Vectors (Plasmids / Cosmids):
Used in microbes for gene cloning and expression.
Example: E. coli plasmid vectors, shuttle vectors.
C. Chemical Methods

Calcium Phosphate Method:
DNA forms a precipitate with CaPO₄, which is taken up by mammalian cells.
Cationic Polymers or Lipids:
DNA binds positively charged molecules to facilitate uptake.
5. Factors Affecting Gene Transfer

Type of target cell: Plant, animal, bacterial, or fungal cells
DNA size and form: Circular plasmid, linear DNA, or RNA
Delivery method: Efficiency varies with method
Promoter and vector choice: Determines expression
Host cell physiology: Actively dividing cells are more receptive.

6. Applications of Gene Transfer

Transgenic Plants:
Traits: Pest resistance, herbicide tolerance, improved nutrition
Examples: Bt cotton, Golden rice
Transgenic Animals:
Study of gene function, disease models, pharmaceutical protein production
Example: Transgenic mice expressing human insulin
Gene Therapy:

Treating genetic disorders by delivering functional genes to patients
Example: SCID, hemophilia
Vaccine Development:
DNA vaccines introduce antigen genes into host cells for immune response
Industrial Biotechnology:
Microbes engineered for enzyme production, biofuels, or pharmaceuticals
7. Advantages of Gene Transfer Technologies
Precise introduction of desired genes
Enables functional genomics and molecular breeding
Production of therapeutic proteins and vaccines
Accelerates trait improvement in plants and animals
Can study gene regulation and protein function
8. Limitations
Low efficiency in some cells or organisms
Risk of insertional mutagenesis (especially with viral vectors)
Immune responses in animals (viral vectors)
Labor-intensive and technically demanding
Regulatory and ethical considerations for human or transgenic applications

Applications

Transgenic plants & animals, gene therapy, vaccines, industrial biotech
Advantages
Precision, functional analysis, trait improvement, protein production
Limitations
Low efficiency, insertional mutagenesis, immune response, technical complexity




Gene Transfer Technologies – 50 MCQs


Gene transfer is defined as:
a) Deleting genes from a genome
b) Introducing foreign DNA into a target organism ✅
c) RNA transcription
d) Protein translation
Which of the following is a natural gene transfer mechanism?
a) Microinjection
b) Transformation ✅
c) Electroporation
d) Lipofection
Which gene transfer method uses a fine glass needle?
a) Electroporation
b) Microinjection ✅
c) Biolistics
d) Agrobacterium-mediated
Particle bombardment is also called:
a) Lipofection
b) Biolistics ✅
c) Microinjection
d) Electroporation
Which bacterium is commonly used for plant gene transfer?
a) E. coli
b) Agrobacterium tumefaciens ✅
c) Bacillus subtilis
d) Pseudomonas
Electroporation involves:
a) Chemical uptake of DNA
b) Electric pulses to create pores ✅
c) Viral infection
d) Microinjection
Which viral vector integrates into the host genome?
a) Adenovirus
b) Retrovirus ✅
c) Baculovirus
d) AAV
Which vector is preferred for transient gene expression?
a) Retrovirus
b) Adenovirus ✅
c) Agrobacterium
d) Plasmid only
Calcium phosphate method is a type of:
a) Physical gene transfer
b) Chemical gene transfer ✅
c) Viral vector
d) Agrobacterium-mediated
Lipofection uses:
a) Lipid vesicles to deliver DNA ✅
b) Electroporation
c) Virus infection
d) Microinjection
Gene transfer in animals can be achieved by:
a) Microinjection ✅
b) Biolistics
c) Agrobacterium
d) Electroporation in plants only
Which plant transformation method is most suitable for dicots?
a) Biolistics
b) Agrobacterium-mediated ✅
c) Electroporation
d) Lipofection
In gene transfer, the foreign DNA can exist as:
a) Integrated into the genome ✅
b) Episomal DNA ✅
c) RNA only
d) Both a and b ✅
Adeno-associated virus (AAV) is preferred because:
a) High immunogenicity
b) Low immunogenicity ✅
c) Integrates randomly
d) Cannot infect mammalian cells
Which method is most commonly used for producing transgenic mice?
a) Electroporation
b) Microinjection ✅
c) Biolistics
d) Agrobacterium-mediated
Advantages of Agrobacterium-mediated transformation include:
a) Random insertion only
b) Stable integration ✅
c) Works only in animals
d) Only transient expression
The gene gun delivers DNA using:
a) Lipid vesicles
b) Helium pressure ✅
c) Viral vectors
d) Electric pulses
Which method is efficient for bacterial gene transfer in lab?
a) Transformation ✅
b) Microinjection
c) Lipofection
d) Biolistics
Which viral vector is non-integrating and used for short-term expression?
a) Retrovirus
b) Adenovirus ✅
c) Lentivirus
d) Plasmid
Chemical-mediated gene transfer includes:
a) Calcium phosphate ✅
b) Electroporation
c) Microinjection
d) Biolistics
In gene therapy, viral vectors are used to:
a) Deliver functional genes ✅
b) Delete chromosomes
c) Transform plants only
d) Measure protein expression
Which gene transfer method is widely used in plant monocots?
a) Agrobacterium
b) Biolistics ✅
c) Microinjection
d) Lipofection
Transformation in bacteria involves:
a) Uptake of naked DNA ✅
b) Viral infection only
c) Microinjection
d) Lipid vesicles
Conjugation in bacteria is mediated by:
a) Plasmids ✅
b) Viruses
c) Liposomes
d) Microinjection
Transduction is gene transfer mediated by:
a) Bacteria
b) Viruses ✅
c) Liposomes
d) Electric pulses
Which method is labor-intensive but precise in animal gene transfer?
a) Electroporation
b) Microinjection ✅
c) Lipofection
d) Agrobacterium-mediated
Biolistics is advantageous because:
a) No tissue limitation ✅
b) Only works in dicots
c) Low DNA delivery
d) Cannot transform plants
Gene transfer efficiency depends on:
a) Vector type ✅
b) Target cell type ✅
c) DNA form
d) All of the above ✅
Shuttle vectors can transfer genes between:
a) Bacteria only
b) Plant and bacteria ✅
c) Animals only
d) Viruses only
Which gene transfer method can cause immune response in animals?
a) Retroviral vector ✅
b) Biolistics
c) Lipofection
d) Microinjection
Stable expression occurs when:
a) DNA integrates into genome ✅
b) DNA remains episomal
c) DNA degrades
d) RNA is used
Transient expression occurs when:
a) DNA integrates
b) DNA remains episomal ✅
c) DNA mutates
d) RNA is degraded
Agrobacterium T-DNA integrates into:
a) Chloroplast DNA
b) Nuclear genome ✅
c) Mitochondrial DNA
d) Cytoplasm
Electroporation is widely used in:
a) Plant protoplasts ✅
b) Animal tissue only
c) Viral vectors
d) Microinjection
Liposome-mediated gene transfer is also called:
a) Electroporation
b) Lipofection ✅
c) Biolistics
d) Agrobacterium method
Viral vectors used in humans include:
a) Retrovirus ✅
b) Adenovirus ✅
c) Lentivirus ✅
d) All of the above ✅
Gene transfer in bacteria can occur naturally through:
a) Transformation ✅
b) Transduction ✅
c) Conjugation ✅
d) All of the above ✅
Calcium phosphate precipitation introduces DNA into:
a) Bacteria
b) Mammalian cells ✅
c) Plant cells only
d) Viruses
Advantages of viral vectors include:
a) High efficiency ✅
b) Targeted delivery
c) Stable or transient expression
d) All of the above ✅
Limitations of gene transfer include:
a) Low efficiency in some cells ✅
b) Insertional mutagenesis ✅
c) Immune response in animals ✅
d) All of the above ✅
DNA form suitable for transformation is:
a) Linear DNA ✅
b) Circular plasmid ✅
c) Both a and b ✅
d) RNA only
Gene transfer is used in industrial biotechnology to:
a) Produce enzymes ✅
b) Produce biofuels ✅
c) Produce pharmaceuticals ✅
d) All of the above ✅
AAV vector advantage is:
a) Integrates randomly
b) Low immunogenicity ✅
c) Only infects plants
d) Cannot deliver DNA
Which plant transformation method uses Agrobacterium Ti plasmid?
a) Biolistics
b) Agrobacterium-mediated ✅
c) Electroporation
d) Lipofection
Microinjection is mainly used for:
a) Plant cells only
b) Animal embryos ✅
c) Bacteria only
d) Viruses only
Particle bombardment is effective in:
a) Plants ✅
b) Mammalian cells
c) Bacteria
d) Viruses
Transient expression is useful for:
a) Short-term studies ✅
b) Long-term gene therapy
c) Permanent genome alteration
d) Cloning DNA
Shuttle vectors allow gene transfer between:
a) E. coli and yeast ✅
b) Plant and animal
c) Virus and bacteria
d) All of the above
Factors affecting gene transfer include:
a) DNA size ✅
b) Promoter strength ✅
c) Host cell type ✅
d) All of the above ✅
Gene transfer technology applications include:
a) Transgenic plants ✅
b) Transgenic animals ✅
c) Gene therapy ✅
d) All of the above ✅

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