Lentiviral Production

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Introduction.

Lentiviral vector production is complex. Non viral expression plasmids are produced which encode the structural components of the virus and the packaging elements required to package the viral single stranded RNA into the final vector. The entire production process takes place inside human cells grown in culture.

 

Transfection of Expression Plasmids.

The plasmids encoding the viral coat proteins, the virion components and the genome plasmid are transfected into human cell culture. They are imported into the cell nucleus and start expressing the genes they encode.

LV_Production_1.jpg

 

Envelope Proteins.

The envelope proteins direct the tissue specificity of the lentiviral vector. In the case of our work we express modified versions of the Sendai Virus H & FN proteins to try and re-target the lentivirus. However, they are not directly packaged into the vector but are exported to the cell membrane of the production cell.

LV_Production_2.jpg

Changing a Virus Pseudotype.

Lentivirus Animation
Basic description of Lentivirus Pseudotyping.

 

Virion Packaging.

The virion proteins and produced and remain within the cell. The LV genome is single stranded RNA which is produced from the genome plasmid. The ssRNA genome is packaged into the virion.

LV_Production_3.jpg

 

Budding of the Virion.

The viral virions fuse with the cell membrane of the production cell and bud off into the cell culture medium. This process ensures that the final vectors and surrounded by a lipid bilayer which contains the envelope proteins.

LV_Production_4.jpg

 

Vector Purification.

Various filtration and purification methods are then used to process the cell culture media. This produces a final vector fraction free of contaminants, other cell products or the plasmid molecules.


 

Light microscope image of a human airway liquid interface cultures. Dark patches are mucous.

 

Large scale lentivirus production in suspension culture.

 

A CFTR Western blot, to confirm protein production in cell culture.

 

Mouse lung large airway (cell nuclei blue) transduced with an adenoviral vector (green).