Gene Therapy and Viral Vectors 2

ends lecture 10 slide 6

Herpes Simplex Virus: Classification and Core Properties

• Family: Herpesviridae (α-herpesvirus subgroup)

• Genome type: Linear double-stranded DNA (dsDNA)

• Genome size: ~150–200 kb (HSV-1 ≈ 152 kb)

• One of the largest genomes among human viruses used in gene therapy

Herpes Simplex Virus: Diagram

Structural Components of Herpes Simplex Virus

1. Core

2. capsid (Icosahedral protein shell)

3. Tegument

4. Envelope (lipid bilayer + glycoproteins)

Structural Components of Herpes Simplex Virus: Core

• Contains linear dsDNA genome (~152 kb)

• DNA is tightly packed under high internal pressure inside capsid

• Delivered directly into nucleus through nuclear pore complex

Functional implication

• Rapid genome release → immediate access to host transcription machinery

• No need for cytoplasmic replication steps

Structural Components of Herpes Simplex Virus: Capsid (Icosahedral protein shell)

• Surrounds and protects viral DNA

• Structure: icosahedral symmetry (T=16)

Depth

• Composed mainly of:

  • Major capsid protein VP5

• Highly stable:

  • Protects genome during:

    • Extracellular transmission

    • Intracellular transport

Mechanistic role

• Travels along microtubules (dynein-mediated) to nucleus

• Docking at nuclear pore → DNA injection

Structural Components of Herpes Simplex Virus: Tegument

• Protein layer between capsid and envelope

• Contains regulatory viral proteins

• Major tegument proteins:

  • VP16 → activates immediate-early (α) gene transcription

  • VHS (virion host shutoff protein) → degrades host mRNA

Functional consequences

• Virus controls host cell immediately upon entry:

  • Shuts down host protein synthesis

  • Redirects machinery toward viral gene expression

Structural Components of Herpes Simplex Virus: Envelope (lipid bilayer + glycoproteins)

• Derived from host membranes

• Embedded with ~10 viral glycoproteins

Key glycoproteins

• gB, gC → initial attachment (heparan sulfate binding)

• gD → receptor engagement (HVEM, nectin)

Depth

• Entry mechanism:

  • Membrane fusion, not endocytosis (in many cells)

• This allows:

  • Direct release of capsid into cytoplasm

Functional implication

• Determines:

  • Cell tropism (which cells can be infected)

functions of tegument in HSV

• Initiates viral gene expression

  • VP16 activates Immediate Early (α) genes → rapid transcription onset

• Shuts off host protein synthesis

  • VHS protein degrades host mRNA → shifts translation to viral proteins

• Enables capsid transport to nucleus

  • Interacts with dynein/microtubules → efficient delivery to nuclear pore

• Suppresses host immune response

  • Interferes with interferon signaling → delays detection

• Supports virion assembly

  • Acts as a bridge between capsid and envelope during maturation

Synthesis: Tegument proteins allow HSV to immediately control host processes, ensuring rapid gene expression, efficient genome delivery, and evasion of early immune responses.

HSV glycoprotein function

• Initial attachment to host cells

  • gB and gC bind heparan sulfate on cell surface → concentrates virus on membrane

• Receptor recognition and entry specificity

  • gD binds entry receptors (e.g., HVEM, nectin) → determines cell tropism

• Membrane fusion and viral entry

  • gD activation triggers gB + gH/gL fusion machinery

  • Leads to fusion of viral envelope with host membrane → capsid released into cytoplasm

• Cell-to-cell spread

  • Glycoproteins mediate fusion between infected and adjacent cells

  • Enables spread without exposure to extracellular immune defenses

HSV Genome Structure Diagram

HSV Genome size and composition

• HSV-1 genome: ~152 kb dsDNA

• Encodes: ~84 viral genes

Depth

• This is a high coding capacity genome, allowing:

  • Structural proteins

  • Enzymes

  • Regulatory proteins

• Compared to smaller viruses, HSV encodes more of its own replication machinery, reducing dependence on host

HSV: Essential vs non-essential genes

• ~50% genes are:

  • Essential → required for viral replication

• Remaining genes:

  • Non-essential (“accessory”)

Depth

• Essential genes include:

  • DNA polymerase

  • Helicase-primase complex

• Non-essential genes mainly:

  • Modify host environment rather than replication itself

HSV: functions of non-essential genes

These genes are important for:

• Immune evasion

• Replication in non-dividing cells

• Shutdown of host protein synthesis

Depth

• Immune evasion:

  • Inhibits interferon signaling

• Non-dividing cell replication:

  • Critical for infection of neurons

• Host shutoff:

  • Ensures viral dominance over cellular machinery

HSV: Gene deletion and vector design

• Non-essential genes can be:

  • Deleted

  • Replaced with exogenous (therapeutic) DNA

Depth (this is the key concept)

• Common deletions:

  • ICP34.5 → reduces neurovirulence

  • ICP4 → blocks replication (creates replication-deficient vector)

Result

• Space created for:

  • Large therapeutic inserts (~30–50 kb)

HSV Epidemiology: Classification within α-herpesviruses

• HSV belongs to alphaherpesviruses (α-HVs)

• Human α-HVs include:

  • HSV-1

  • HSV-2

  • Varicella Zoster Virus

Depth

• α-herpesviruses are characterized by:

  • Rapid replication (lytic phase)

  • Ability to establish latent infection in sensory neurons

HSV Epidemiology: Transmission and disease types

• HSV-1

  • Transmitted via oral contact

  • Causes oral herpes (cold sores)

• HSV-2

  • Sexually transmitted

  • Causes genital herpes

Depth

• Both viruses:

  • Infect epithelial cells initially

  • Then establish latency in neurons

• HSV-1 can also cause genital infections (increasingly common)

HSV Epidemiology: Global prevalence

• HSV-1:

  • ~3.7 billion people (<50 years) → ~67% infected

• HSV-2:

  • ~491 million (15–49 years) → ~13% infected

Depth (important implication)

• Extremely high prevalence → many individuals have:

  • pre-existing immunity

HSV: Clinical presentation

• Most infections:

  • Asymptomatic

• When symptomatic:

  • Painful blisters or ulcers

Depth

• Virus replication causes:

  • Cell lysis → tissue damage → lesions

• Immune response contributes to:

  • Inflammation and pain

HSV: Associated diseases

• HSV infections linked to:

  • Oral/genital herpes

• Varicella Zoster Virus causes:

  • Chickenpox

Depth

• Important distinction:

  • HSV → recurrent localized lesions

  • VZV → systemic infection (chickenpox) + later reactivation (shingles)

HSV Clinical Manifestations:

HSV Clinical Manifestations: Neurotropic manifestations

• Encephalitis (HSV-1)

• Meningitis (HSV-1 > HSV-2)

• Mollaret’s meningitis (HSV-2)

• Bell’s palsy (HSV-1)

Depth

• HSV has strong neurotropism → infects sensory neurons

• Travels via:

  • retrograde axonal transport → CNS

• HSV encephalitis:

  • Typically affects temporal lobe

  • Due to viral replication + immune-mediated damage

HSV Clinical Manifestations: Ocular infections

• Keratitis (HSV-1)

• Conjunctivitis (HSV-1)

• Retinitis (HSV-2)

Depth

• Keratitis is:

  • One of the leading causes of infectious blindness

• Mechanism:

  • Viral replication damages corneal epithelium

  • Recurrent infections worsen damage due to immune scarring

HSV Clinical Manifestations: Oral and facial infections

• Oral herpes (labialis) — HSV-1 > HSV-2

• Gingivostomatitis — HSV-1

Depth

• Primary infection:

  • Often gingivostomatitis (severe, widespread lesions)

• Reactivation:

  • Localized cold sores

• Occurs due to:

  • Reactivation from trigeminal ganglion latency

HSV Clinical Manifestations: Skin infections

• Eczema herpeticum (HSV-1)

• Herpes gladiatorum (HSV-1)

• Herpetic whitlow (HSV-1)

Depth

• Occur when virus enters through:

  • broken skin barrier

• Gladiatorum:

  • Seen in contact sports (skin-to-skin transmission)

• Whitlow:

  • Infection of fingers (common in healthcare workers)

HSV Clinical Manifestations: Genital infections

• Genital herpes

  • Primary: HSV-1 > HSV-2

  • Recurrent: HSV-2 > HSV-1

Depth (important distinction)

• HSV-2:

  • Better adapted to genital tract latency

  • Causes more frequent reactivation

• HSV-1:

  • Increasing cause of primary genital infections

  • But less recurrent

Life Cycle of Herpes Simplex Virus: Attachment and Entry

• Viral glycoproteins:

  • gB, gC → bind heparan sulfate

  • gD → binds entry receptors (HVEM, nectin)

• Triggers:

  • gB + gH/gL–mediated membrane fusion

Outcome

• Viral envelope fuses with host membrane

• Capsid + tegument released into cytoplasm

Life Cycle of Herpes Simplex Virus: Capsid Transport to Nucleus

• Capsid moves via:

  • microtubules (dynein-mediated retrograde transport)

Outcome

• Capsid docks at:

  • nuclear pore complex

• Viral DNA is:

  • injected into nucleus

Life Cycle of Herpes Simplex Virus: Immediate Host Takeover (Tegument action)

• VP16 → activates α (immediate early) genes

• VHS → degrades host mRNA

Outcome

• Rapid shift from:

  • host → viral gene expression

Life Cycle of Herpes Simplex Virus: Transcriptional Cascade

Three phases:

1. α (Immediate Early)

   • Regulatory proteins

2. β (Early)

   • DNA replication enzymes

3. γ (Late)

   • Structural proteins

Outcome

• Controlled, sequential gene expression

Life Cycle of Herpes Simplex Virus: Viral DNA Replication

• Genome:

  • Linear → circularizes in nucleus

Replication mechanism:

• Starts as theta replication

• Switches to rolling circle replication

Outcome

• Formation of:

  • concatemeric DNA (long repeats)

Life Cycle of Herpes Simplex Virus: Assembly

• Capsid assembly:

  • Occurs in nucleus

• Viral DNA:

  • Packaged into capsid

Tegument addition:

• Occurs during:

  • cytoplasmic transit

Life Cycle of Herpes Simplex Virus: Envelopment and Release

• Virus acquires envelope by:

  • budding through nuclear membrane

Outcome

• Mature virions transported via:

  • vesicles → released by exocytosis

HSV: Lytic vs Latent Pathways

Lytic cycle

• Active replication

• Cell lysis → virus release

Latent cycle

• Viral DNA persists as:

  • episome in sensory neurons

• Only:

  • LAT expressed

Reactivation

• Triggered by stress, immunosuppression

• Virus travels:

  • anterograde → epithelial cells → re-infection

HSV lifecycle overview + diagram

• Attachment and Entry

• Capsid Transport to Nucleus

• Immediate Host Takeover

• Transcriptional Cascade

• Viral DNA Replication

• Assembly

• Envelopment and Release

All α-HVs initially infect _______ cells (primary site of infection), and later spread to infect ________

epithelial

sensory neurons.

HSV as a Latent Neurotropic Virus: Acute Infection

• Infection begins in peripheral epithelial cells

What’s happening mechanistically

• Virus undergoes lytic replication:

  • Produces virions

  • Causes epithelial cell death

• Leads to:

  • Cold sores

  • Viral shedding (transmission stage)

HSV as a Latent Neurotropic Virus: Establishment of Latency

• Virus enters sensory neurons

• Moves via:

  • Retrograde axonal transport → trigeminal ganglia

Inside neuron

• Viral DNA:

  • Circularizes → episomal form

• Transcription is largely silenced except:

  • LAT (Latency-Associated Transcript)

Functional role of LAT

• Suppresses:

  • Viral lytic gene expression

• Prevents:

  • Apoptosis of infected neuron
    → Ensures long-term persistence

HSV as a Latent Neurotropic Virus: Reactivation

• Triggered by:

  • Stress, UV light, immunosuppression

Mechanism

• Viral genome reactivates:

  • Lytic genes expressed again

• Virus travels via:

  • Anterograde transport → back to epithelial cells

HSV as a Latent Neurotropic Virus: Secondary Infection / Recurrence

• Virus reaches epithelial cells again

Outcomes (from slide)

• Cold sores

• Viral shedding

• Epithelial cell death

Key insight

• Recurrence occurs at:

  • same anatomical site

  • Due to fixed neuronal reservoir

HSV as a Latent Neurotropic Virus: CNS involvement

Possible outcomes:

• Herpes simplex encephalitis

• Neuronal cell death

• Associations with:

  • Multiple sclerosis

  • Alzheimer’s disease

Depth

• Occurs when virus spreads beyond peripheral neurons into:

  • central nervous system

• HSV-1 encephalitis:

  • Often targets temporal lobe

HSV as a Latent Neurotropic Virus Diagram

Challenges of HSV as a Gene Therapy Vector

• Immunogenicity

• Packaging Constraints

• Random Integration

• Cytotoxicity / Lytic Nature

Challenges of HSV as a Gene Therapy Vector: Immunogenicity

• HSV particles trigger a strong immune response

Mechanistic depth

• Viral proteins (especially envelope glycoproteins + tegument proteins) are recognized by:

  • Innate immunity (TLRs, interferon response)

  • Adaptive immunity (neutralizing antibodies, T cells)

Consequences

• Rapid vector clearance

• Reduced transgene expression duration

• Difficulty with repeat dosing

Challenges of HSV as a Gene Therapy Vector: Packaging Constraints

• Despite large genome, there are limits to how much DNA can be inserted

Mechanistic depth

• Capsid has a physical size limit → cannot exceed stable genome length

• Overloading genome:

  • Disrupts capsid assembly

  • Reduces viral stability

Practical implication

• Although HSV can carry ~30–50 kb inserts:

  • Insert size must be balanced with essential genome elements

Challenges of HSV as a Gene Therapy Vector: Random Integration

• HSV DNA is mainly episomal but can rarely integrate into host genome

Mechanistic depth

• Integration may occur via:

  • host DNA repair pathways (non-homologous recombination)

Risks

• Insertional mutagenesis:

  • Disruption of host genes

  • Potential activation of oncogenes

Challenges of HSV as a Gene Therapy Vector: Cytotoxic

• HSV naturally undergoes lytic replication

Depth:

• Causes host cell death

• Problem for:

  • Non-cancer gene therapy

• Requires:

  • attenuation (e.g., ICP34.5 deletion)

Benefits of Herpes Simplex Virus as a Vector

• Broad Cell Tropism

• Natural Cytolytic Activity

• Large Genome → Gene Insertion Capacity

• Engineering Flexibility

• Synergy with Other Therapies

• Episomal Persistence

Benefits of Herpes Simplex Virus as a Vector: Broad Cell Tropism

• HSV can infect a wide variety of cell types

Depth

• Due to multiple entry receptors (heparan sulfate, HVEM, nectins)

• Infects:

  • Dividing cells (tumors)

  • Non-dividing cells (neurons)

Why this is impressive

• Many vectors (e.g., retroviruses) cannot infect non-dividing cells
  → HSV is versatile across tissues

Benefits of Herpes Simplex Virus as a Vector: Natural Cytolytic Activity

• HSV replication leads to cell lysis

Depth

• Viral replication:

  • Disrupts cellular machinery

  • Causes membrane breakdown

Application

• Direct killing of:

  • Cancer cells (oncolysis)

Benefits of Herpes Simplex Virus as a Vector: Large Genome → Gene Insertion Capacity

• Contains many non-essential genes

Depth

• These can be:

  • Deleted

  • Replaced with therapeutic genes

Outcome

• Can insert:

  • Large or multiple genes (~30–50 kb)

• Supports:

  • Complex therapies (e.g., gene + regulatory elements)

Benefits of Herpes Simplex Virus as a Vector: Engineering Flexibility

• HSV can be re-engineered

From slide

• Can express:

  • Cytotoxic genes

  • Immune-stimulating genes

Depth

• Examples:

  • Prodrug-activating enzymes

  • Cytokines (e.g., GM-CSF)

• Enables:

  • targeted tumor destruction + immune activation

Benefits of Herpes Simplex Virus as a Vector: Synergy with Other Therapies

• Works well with:

  • Radiation therapy

  • Chemotherapy

Depth

• Viral infection can:

  • Increase tumor sensitivity to radiation

  • Enhance immune-mediated tumor clearance

Benefits of Herpes Simplex Virus as a Vector: Episomal Persistence

• HSV genome remains episomal (non-integrating)

Why it matters

• Reduces:

  • Insertional mutagenesis risk

• Supports:

  • Safer gene delivery

Major Types of Cancer

-Carcinoma is a cancer that begins in the skin or in tissues that line or cover internal organs.

• Sarcoma is a cancer that begins in bone, cartilage, fat, muscle, blood vessels, or other connective or supportive tissue.

• Leukemia is a cancer that starts in blood-forming tissue, such as the bone marrow, and causes large numbers of abnormal blood cells to be produced and enter the blood.

• Lymphoma and multiple myeloma are cancers that begin in the cells of the immune system.

• Central nervous system cancers are cancers that begin in the tissues of the brain and spinal cord.

“Drivers” of Cancer: proto-oncogenes

1. Proto-oncogenes → Oncogenes (from slide)

• Normal role: promote cell growth and division

• When altered → become oncogenes

Mechanism

• Gain-of-function mutations:

  • Overexpression

  • Constitutive activation

Examples (from slide)

• HER2, Ras, Myc

Functional outcome

• Cells:

  • Proliferate without external growth signals

  • Avoid normal growth limits

“Drivers” of Cancer: tumor suppressor genes

• Normal role: inhibit cell division / control cell cycle

Mechanism

• Loss-of-function mutations:

  • Remove growth inhibition

  • Disable cell cycle checkpoints

Examples (from slide)

• p53, p10

Functional outcome

• Cells divide:

  • Uncontrollably

  • Even when damaged

“Drivers” of Cancer: DNA repair genes

• Normal role: repair damaged DNA

Mechanism

• Mutation → defective repair system

• Leads to:

  • Accumulation of mutations

Examples (from slide)

• BRCA1, BRCA2

Functional outcome

• Genomic instability:

  • Accelerates cancer progression

Stages of Cancer

1. Purpose of Staging (from slide)

• Determines:

  • Location of cancer

  • Extent of spread

  • Impact on other body parts

Depth

• Staging is not just descriptive — it directly guides:

  • Treatment selection

  • Prognosis estimation

2. Role in Treatment Planning (from slide)

• Helps decide:

  • Surgery (localized tumors)

  • Chemotherapy (systemic disease)

  • Radiation therapy (targeted control)

Depth

• Early-stage:

  • Often treated with localized therapies

• Advanced-stage:

  • Requires systemic approaches

3. Predicting Recurrence (from slide)

• Indicates:

  • Likelihood cancer will return after treatment

Depth

• Higher stage → higher chance of:

  • Residual disease

  • Metastasis

4. Predicting Survival / Recovery (from slide)

• Used to estimate:

  • Patient prognosis

Depth

• Lower stage:

  • Better survival rates

• Higher stage:

  • Poorer outcomes due to spread

5. Standardized Communication (from slide)

• Provides a common language for:

  • Doctors

  • Researchers

Depth

• Ensures:

  • Consistency across hospitals and studies

• Enables:

  • Accurate comparison of patient outcomes

TNM: — Primary Tumor Size & Extent

• Answers:

  • How large is the tumor?

  • Where is it located?

Depth

• T staging reflects:

  • Tumor size (mm/cm)

  • Degree of local invasion into surrounding tissue

• Higher T → greater:

  • Local tissue damage

  • Surgical difficulty

TNM: Node — Lymph Node Involvement

• Answers:

  • Has cancer spread to lymph nodes?

  • If yes: how many and where?

Depth

• Lymphatic spread is often the first route of metastasis

• More nodes involved → higher likelihood of:

  • Systemic dissemination

• Regional lymph nodes act as:

  • checkpoint for cancer spread

TNM: Metastasis (M) — Distant Spread

• Answers:

  • Has cancer spread to other parts of the body?

  • If yes: where and how much?

Depth

• Indicates spread via:

  • blood (hematogenous)

  • or advanced lymphatic spread

• Presence of metastasis (M1):

  • Automatically indicates advanced-stage cancer

TNM Stage grouping: Stage Grouping + diagram

• Stage 0:

  • Abnormal cells, no invasion

• Stage I:

  • Early, localized spread

• Stage II:

  • Larger tumor ± limited lymph node involvement

• Stage III:

  • Extensive regional spread (more lymph nodes, larger tumor)

• Stage IV:

  • Distant metastasis

Treatment of cancer

Surgery

• Chemotherapy

• Radiation Therapy

• Immunotherapy (Oncolytic viral therapy)

• Stem Cell Transplant

• Hyperthermia

• Gene Therapy

Oncolytic HSV Mechanism: Infection of Tumor Cells

• Oncolytic HSV infects tumor cells

Depth

• Tumor cells often have:

  • Defective antiviral responses (e.g., interferon pathway)

• This makes them:

  • More permissive to viral entry and replication

Oncolytic HSV Mechanism: Viral Replication in Tumor Cells

• Virus replicates efficiently inside tumor cells

Depth

• Cancer cells:

  • Have high metabolic activity

  • Provide resources for rapid viral genome replication

• Engineered HSV strains:

  • Preferentially replicate in abnormal cells

Oncolytic HSV Mechanism: Tumor Cell Lysis

• Infected tumor cell ruptures (lysis)

Depth

• Lysis results from:

  • Accumulation of viral particles

  • Breakdown of cellular integrity

Outcome

• Cell death + release of:

  • new virions

Oncolytic HSV Mechanism: Viral Spread

• Released virions infect neighboring tumor cells

Depth

• Creates a self-amplifying cycle:

  • Infection → replication → lysis → spread

• Allows:

  • Progressive destruction of tumor mass

Oncolytic HSV Mechanism: Effect on Healthy Cells

• Virus:

  • Does not replicate efficiently in healthy cells

Depth

• Normal cells:

  • Have intact antiviral defenses

  • Activate interferon pathways → inhibit viral replication

Outcome

• Healthy cells:

  • Survive infection or clear virus

Oncolytic HSV Mechanism diagram HF10 project

Oncolytic virus

An oncolytic virus is a virus that preferentially infects and kills cancer cells. As the infected cancer cells are destroyed by oncolysis, they release new infectious virus particles or virions to help destroy the remaining tumour.

Oncolytic virotherapy

Clinical Trial Phases

1. Phase I — Safety (from slide)

• Purpose: Check for safety

• Sample: 10–20 healthy volunteers

Depth

• Determines:

  • Maximum tolerated dose (MTD)

  • Dose-limiting toxicities

• Unexpected side effects:

  • Common at this stage

2. Phase II — Efficacy (from slide)

• Purpose: Check for efficacy

• Sample: ~200 patients

Depth

• Evaluates:

  • Does the treatment actually work?

• Many treatments fail here because:

  • Effectiveness is lower than expected

3. Phase III — Large-scale confirmation (from slide)

• Purpose: Confirm findings in large population

• Sample: >1000 people

Depth

• Compares:

  • New treatment vs standard therapy/placebo

• Detects:

  • Rare side effects (due to large sample size)

4. Phase IV — Post-marketing surveillance (from slide)

• Purpose: Long-term safety in real-world population

• Sample: General patient population

Depth

• Conducted after:

  • Drug approval

• Identifies:

  • Rare or delayed adverse effects

  • Effects in:

    • Previously untested groups

Table of properties for viruses

Delivery methods

• Micro-injection

• Electroporation

• Gene gun

• Tattooing

• Laser

• Ultrasound

Electroporation

What it is:
Application of short electrical pulses to create temporary pores in the cell membrane.

Technical details:

• Electric field disrupts lipid bilayer → transient permeability

• DNA enters through these pores

Key features:

• Works on many cells at once

• Efficiency depends on voltage, pulse duration

Exam insight:
Widely used for bacteria, mammalian cells, and in vivo gene delivery

Gene Gun (Biolistic method)

What it is:
DNA-coated metal particles (gold/tungsten) are shot into cells at high velocity.

Technical details:

• Physical penetration delivers DNA directly into cytoplasm/nucleus

• Often used for plant cells (cell wall barrier)

Key features:

• No need for vectors

• Can target tissues directly

Limitation:
Cell damage + shallow penetration

Tattooing

What it is:
Uses rapid needle punctures (like a tattoo machine) to deliver DNA into skin.

Technical details:

• Creates micro-injuries → enhances DNA uptake

• Often used in DNA vaccines

Key features:

• Simple, low-cost

• Works well for skin immune responses

Laser

What it is:
Laser creates temporary holes in cell membranes.

Technical details:

• Highly controlled, localized membrane disruption

• DNA diffuses into cell after pore formation

Key features:

• Precise targeting

• Requires specialized equipment

Ultrasound

What it is:
Uses sound waves (sonoporation) to increase membrane permeability.

Technical details:

• Often combined with microbubbles

• Cavitation effect → membrane disruption

Key features:

• Non-invasive

• Can target deep tissues

Microinjection diagram

What microinjection actually does (mechanism)

• A glass micropipette (~0.5–1 µm tip) physically penetrates the cell membrane

• DNA is directly deposited into:

  • Cytoplasm or

  • Nucleus (more effective for expression)

Key point:
No reliance on endocytosis → no degradation in endosomes/lysosomes

Microinjection Target cells

Slide mentions:

• Eggs

• Oocytes

• Embryos

• Plant protoplasts

Why these?

• Large size → easier to inject

• Visible nucleus → precise targeting

• Protoplasts lack cell wall → easier penetration

Microinjection Equipment

Slide mentions:

• Specialised microscope

• Manipulator

• Phase-contrast microscope

Expanded breakdown:

• Micromanipulator → controls needle movement in micrometers

• Holding pipette → stabilizes the cell using suction

• Injection needle → delivers DNA

• Phase-contrast microscope → allows visualization of transparent cells

Computerized control (from slide):

• Improves:

  • Accuracy

  • Reproducibility

  • Speed

• Video systems help monitor injection in real time

Role of dye in microinjection

• Dye is co-injected with DNA

• Helps identify:

  • Whether injection was successful

  • Which cells received DNA

Exam angle:
Acts as a visual marker, not functional in gene expression

microinjections limitations

• Very low throughput (one cell at a time)

• Requires high technical skill

• Risk of cell damage or lysis

• Expensive equipment

Microinjection: ideal cell characteristics

• Large size

• Non-adherent

• Pronounced nucleus

Why these matter (technical reasoning)

• Large cells → easier needle insertion, lower chance of rupture

• Non-adherent cells → easier to manipulate and position under microscope

• Pronounced nucleus → allows accurate nuclear injection, which increases gene expression efficiency

Microinjection problematic cell types

• Contractile cells (e.g., muscle)

• Monolayer adherent cells

Why they are difficult

Contractile cells:

• Rapid shape change during injection

• Calcium influx triggers contraction → needle displacement → cell damage

Adherent cells (monolayer):

• Attached to surface → harder to position and stabilize

• Needle insertion angle becomes difficult

• Increased mechanical stress during injection

DNA Injection (Pronuclear stage)

• Following fertilization, male and female pronuclei remain separate for a few hours before fusion

• This allows microinjection of desired genes into the larger male pronucleus

Technical integration:

• Male pronucleus = larger + more visible → easier targeting

• Injection at this stage ensures DNA is present before first mitotic division

• Increases chance of genome-wide distribution of transgene

DNA Injection (Site of injection)

• Injection is performed into the male pronucleus

Technical integration:

• Nuclear injection avoids cytoplasmic degradation

• Promotes random integration into host genome

• Higher efficiency than cytoplasmic delivery

DNA Injection (Embryo survival and transfer)

• Eggs that survive injection are transferred into oviducts of a pseudopregnant female mouse

Technical integration:

• Pseudopregnancy = hormonally prepared uterus (via mating with vasectomized male)

• Provides:

  • Proper implantation environment

  • Normal embryonic development conditions

DNA Injection (Founder mouse generation)

• Leads to generation of a Founder mouse, from which permanent transgenic lines can be established

Technical integration:

• Founder = organism with integrated transgene

• Used for breeding → stable inheritance (germline transmission)

• Not all offspring are transgenic due to:

  • Random integration

  • Possible mosaicism

DNA Injection (Detection of transgene)

• Presence identified by:

  • PCR analysis

  • Southern blot hybridization

Technical integration:

• PCR → fast detection of gene presence

• Southern blot → confirms:

  • Integration into genome

  • Copy number

  • Insertion pattern

DNA Injection steps

1. Fertilization → pronuclei visible

2. Inject DNA into male pronucleus

3. Select surviving embryos

4. Transfer to pseudopregnant female

5. Birth of offspring

6. Screen using PCR/Southern blot

7. Identify founder → establish transgenic line

DNA injection diagram

Applications: Transgenic Animals (Fertile mating → one-cell embryos)

• Fertile male × female → zygote formation

• Collection of one-cell embryos (early stage)

Technical integration:

• One-cell stage = ideal for genetic modification

• Ensures any inserted DNA can propagate to all cells (germline inclusion)

Applications: Transgenic Animals (Injection of transgene into male pronucleus)

• Transgene is injected into male pronucleus

Technical integration:

• Male pronucleus:

  • Larger → easier targeting

  • More transcriptionally active early on

• DNA integrates randomly into genome

• Occurs before first division → increases stable inheritance

Applications: Transgenic Animals (Embryo implantation)

• Injected embryos are implanted into pseudopregnant females

Technical integration:

• Pseudopregnant female = hormonally primed uterus

• Required because:

  • Embryos cannot develop ex vivo

• Implantation site: oviduct/uterus

Applications: Transgenic Animals (Use of sterile male)

• Female is mated with sterile (vasectomized) male

Technical integration:

• Triggers:

  • Hormonal changes (progesterone increase)

  • Uterine receptivity

• Ensures no competing fertilization

Applications: Transgenic Animals (Live birth + testing)

• Offspring are born → tested for transgene

Technical integration:

• Not all offspring are transgenic

• Screening methods:

  • PCR → presence of gene

  • Southern blot → integration pattern

Applications: Transgenic Animals (Transgenic founder animal)

• Positive offspring = Transgenic founder

Technical integration:

• Founder carries gene in germline

• Can be bred → stable transgenic lineage

• Important for:

  • Functional gene studies

  • Disease models

  • Drug testing