You've been working hard packaging and harvesting your lentiviruses,
Now the most exciting day has finally arrived.
The day you use your lentiviruses to infect your target cells!
In this video, we will take you through:
1. How to perform a lentiviral transduction experiment
2. An overview of the spinoculation and reverse transduction methods
3. How to confirm transgene expression
The recombinant lentivirus is an incredibly useful tool for delivering genes to not only dividing cells,
But also post-mitotic, non-dividing cells like neurons.
To learn more about how the lentivirus system works,
Watch our video, "The Basics of the Recombinant Lentivirus System".
In a nutshell, lentiviruses express two enzymes that enable them to integrate genes into a host cell.
A reverse transcriptase which converts viral RNA into double-stranded viral DNA.
An integrase which enables its viral DNA to be inserted into the host cell's DNA.
Using a recombinant lentivirus, you can deliver cDNA, shRNA, and sgRNA to stably express,
Knockdown, or knockout your gene of interest in your target cells.
Depending on the cell type you are working with, a different transduction protocol is recommended!
Today, we will mainly focus on transduction into adherent cells
And briefly introduce the spinoculation method and the reverse transduction method,
Two methods that tackle suspension cells and difficult-to-transduce cells.
Before you start, ensure your lentiviral construct delivers antibiotic resistance or expresses a fluorescent marker.
This will enable you to isolate your successfully infected cells from the rest of the cell population.
OK, let's go over the general protocol!
Preparing Your Target Cells
24 hours before infection,
Plate your cells at a seeding density of 6-8 x 10⁴ cells/well in a 24-well plate using 1ml of Complete Media.
If you're using a different plate size,
Use a seeding density that ensures that your cells are 30-40% confluent at the time of infection.
Incubate the cells overnight at the appropriate temperature and CO₂ conditions.
Infecting Your Target Cells
Prepare a mixture of Complete Culture Media and Polybrene at a concentration of 8 μg/ml.
Polybrene is a cationic polymer that neutralizes repulsive charges
between the virus and cell surfaces to increase transduction efficiency.
But be careful!
Excessive exposure can cause cytotoxicity.
Remove the growth media in each well and replace with 1ml of the mixture per well
or adjust volume as necessary if you are using a different plate size.
If transduction efficiency is low or if polybrene is toxic to your target cells,
Add ViralEntry™ Transduction Enhancer (Cat. No. G515) instead of polybrene at a ratio of 1:100.
ViralEntry™ has been shown to boost viral transduction up to 10X!
For more information, visit the link provided in the video description.
Prepare the culture wells with the following controls:
A positive control well containing a GFP control lentivirus and an appropriate blank control lentivirus
A negative control well containing uninfected cells.
Based on the optimal MOI that you have calculated,
Use the appropriate volume of virus to infect the cells in the wells.
For more details on how to determine the MOI for your infection experiment,
Watch our video, "Multiplicity of Infection (MOI): What is it and how do I calculate it?"
Which you can find a link for in the video's description below.
Incubate the cells with the virus overnight at the appropriate temperature and CO₂ conditions
Stop Transduction
Remove the culture medium containing the virus and replace with 1ml of fresh Complete Culture Media.
Incubate cells at the appropriate temperature and CO₂ conditions overnight.
Observe Cell Growth
Depending on the growth rate of your target cells, split the cells at a ratio of 1:3 or 1:5.
Continue incubation for another 48 hours in Complete Culture Media.
If your lentivirus carries a fluorescent marker,
You can check for successful infection under a fluorescent microscope.
Select for Successfully Transduced Cells
Successfully infected cells can be selected by adding the minimum concentration of antibiotic needed
to kill untransduced cells, as determined by a killing curve.
At this time, it is recommended to maintain antibiotic selection until the control (untransduced) cells completely die
and antibiotic-resistant colonies become visible.
After this, you may reduce or remove the antibiotic from the culture medium.
If you're infecting suspension cells such as T or B cells,
Or if you're working with hard-to-transduce cell types,
You can also try the following two transduction methods:
The Spinoculation Method
The Reverse Transduction Method
The Spinoculation Method
The Centrifugal Inoculation or Spinoculation transduction method involves an additional centrifugation step.
The additional centrifugal force assists the binding of viruses to cells and improves overall transduction efficiency.
You can access our detailed Spinoculation Protocol in the video description.
The Reverse Transduction Method
Another method that can improve overall transduction efficiency is called the Reverse Transduction method.
In this method, viral particles are first coated onto the culture vessel surface before the cells are added.
This allows for transduction of cells during cell attachment.
This method is commonly used for suspension cells or high throughput applications.
And you've done it!
You've successfully infected your cells!
But how do you know if all your cells have the same mutation and expression profiles?
The answer is, they won't!
At least, not initially.
Confirming Transgene Expression
In lentiviral transduction, gene integration is random,
Meaning that the transgene will be integrated into different sites within the target cell's genome.
Each cell may also have integrated a different number of transgene copies.
All of this results in a heterogenous or polyclonal cell population with varying transgene expression levels.
Depending on the downstream application and research goals,
Subsequent monoclonal selection of cells may be required.
For long term studies, variability issues may also arise
especially when low expressing cell lines have growth advantage over the high expressing ones.
If your goal is simply to confirm whether 100% of your cells have been infected successfully,
You can check for fluorescent marker expression or other expression tags.
To check that a gene knockout or knockdown has been successful,
A Western Blot assaying protein expression would be a better option.
But, if you are working with difficult-to-transduce cells where less than 100% transduction occurs,
You may want to isolate a single cell to expand into a monoclonal cell line.
Or, if you are studying the effects of a gene knockout,
It is imperative that monoclonal cell selection is performed.
You can isolate a homogenous monoclonal cell line
with your ideal transgene expression using the dilution cloning method.
In this method, the polyclonal cells are serially diluted until a single cell remains,
Which is expanded to create your monoclonal cell line.
Alternatively, single-cell sorting methods will also do the trick!
And that's it!
You're now on track to becoming a lentivirus infection expert!
May you have excellent transduction rates in all your experiments!
To learn more on this topic, visit the link to our blog in the video description.
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Thank you for watching!