Viral transduction

HIV-1-derived vectors that integrate into the genome of dividing and non-dividing cells, carry 8–10 kb and give stable expression; made by transient transfection of 293T cells with a split packaging system and usually pseudotyped with VSV-G.

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Lentivirus

How it differs from the standard method

Third-generation systems split the packaging functions across separate plasmids and need no Tat, lowering the chance of a replication-competent recombinant.
Self-inactivating LTRs mean expression comes from an internal promoter such as EF1α, PGK or CMV.
Handle at BSL-2 with institutional approval; integration into the operator's cells is the hazard, not infection.
Concentrate by ultracentrifugation or precipitation when a high MOI or a small volume is needed.
Integration is semi-random with a bias towards active transcription units, so clones differ in expression.

In this lesson · about 3 min read

Borrowing a virus to carry a gene
What the read-out physically is
How it goes wrong
Choosing a vector
Key terms
When to use it
Sub-techniques

1Borrowing a virus to carry a gene

A virus is a machine for getting nucleic acid into a cell. A viral vector keeps that machinery and throws away the part that makes new virus: the genes the particle needs to replicate are removed from the genome it carries and supplied separately, in trans, by the packaging cells.

1 − e^(−MOI)

Poisson sets the ceiling

MOI 0.3 cannot transduce more than about 26% of the cells, however good the vector is.

λ / (1 − e^(−λ))

Copies follow the dose

The copy number in a transduced cell rises with MOI even after the positive fraction has saturated.

TU, not particles

Titre is a measurement

An infectious titre belongs to the cell type, the additive and the day it was measured on.

2What the read-out physically is

Two independent measurements answer two different questions. Flow cytometry for a fluorescent reporter gives the fraction of cells that received and express the vector; it is quick, it needs an untransduced control to set the gate, and before about 72 hours it also reads GFP protein carried in with the vector preparation itself (pseudotransduction), which is not transduction at all.

3How it goes wrong

Calculating the MOI against the number of cells seeded the day before rather than counted on the day.
Repeated freeze-thaw of the vector stock: enveloped vectors lose roughly half their infectivity per cycle.
Quoting a titre measured with polybrene or a spin, then using the vector without them.
Reading a titration from a saturated well, where the positive fraction has stopped tracking the dose.
Chasing 100% transduction with a high MOI, and then interpreting a dose-dependent artefact as a gene function.
Scoring transduction at 48 h, when part of the signal is protein carried in with the prep.
Choosing an AAV serotype by what works in a mouse and expecting it to bind cells on plastic.
Handling lentivector outside BSL-2 containment: the particle integrates into human cells, including yours.

4Choosing a vector

Lentivirus: Integrates, transduces dividing and non-dividing cells, 8–10 kb of cargo, stable expression; BSL-2, and insertional mutagenesis is the standing concern.
γ-Retrovirus: Integrates, but needs the nuclear envelope to break down, so only dividing cells; simpler biology and still used for some cell therapies.
AAV: Stays episomal, very low toxicity and immunogenicity, ~4.7 kb cargo; the dominant in vivo vector, but dosed in genomes per cell in the thousands and slow to express.
Adenovirus: Episomal, very high transduction of almost everything at high MOI, up to ~8 kb (more for gutless vectors), strong immune response in vivo; the usual choice for short, strong expression.

Key terms

Practise these as flashcards →

MOI

Multiplicity of infection: infectious units added per cell present at the time of transduction.

Transducing unit (TU)

A particle that produces a scoreable event in the titration assay used; an operational unit, not a count of particles.

Physical : infectious ratio

Particles (or genomes) per transducing unit; often 100–1000 for lentivector and 10³–10⁴ genomes per transduction for AAV.

Pseudotransduction

Apparent expression from protein or RNA carried inside the vector preparation, which fades over the first two to three days.

Pseudotyping

Putting a different envelope protein (usually VSV-G) on the particle to change its tropism and let it be concentrated by ultracentrifugation.

Vector copy number (VCN)

Integrated vector copies per genome, measured by qPCR or ddPCR against a single-copy reference.

Replication-competent lentivirus (RCL)

A recombinant able to replicate; the contaminant that split packaging systems and self-inactivating LTRs are designed to prevent, and that clinical preparations are tested for.

Self-inactivating (SIN) vector

A design with the promoter-enhancer deleted from the 3′ LTR, so the integrated provirus has no active LTR promoter.

When to use it

To get a construct into primary cells, stem cells or any line that transfects badly.
When expression has to last: an integrating vector gives a population that keeps the cassette through division.
To deliver a library at single copy per cell: the basis of every pooled CRISPR or shRNA screen.
When every cell must express, for example before a selection-free functional assay, using an MOI high enough to saturate.
To express in non-dividing cells such as neurons or differentiated macrophages, where lentivirus and AAV work and retrovirus does not.

Compared with alternatives

Transfection →

Transfection is faster, cheaper and needs no containment; choose it for easy lines, for short transient expression, and for making the vector in the first place.

Stable cell line generation →

Transduction at low MOI followed by selection or sorting is the usual way a stable line is made; the selection step is where single-copy clones are recovered.

CRISPR screens →

Pooled screens depend on this technique's Poisson statistics: a library is transduced at MOI ≈ 0.3 precisely so that most positive cells carry one construct.

RNA interference →

shRNA knockdown is usually delivered as a lentivector, so the dose, copy number and selection arguments here apply to it directly.

Flow cytometry & FACS →

Flow is how transduction efficiency and vector titre are measured, and sorting is how a transduced population is purified when no drug marker is available.

Sub-techniques

Choosing one here switches the whole page, including the protocol and the lab, to it.

Third-generation systems split the packaging functions across separate plasmids and need no Tat, lowering the chance of a replication-competent recombinant.
Self-inactivating LTRs mean expression comes from an internal promoter such as EF1α, PGK or CMV.
Handle at BSL-2 with institutional approval; integration into the operator's cells is the hazard, not infection.
Concentrate by ultracentrifugation or precipitation when a high MOI or a small volume is needed.
Integration is semi-random with a bias towards active transcription units, so clones differ in expression.