On this page

Use the links below to jump to the section you're looking for:

Lentiviral shRNA clones

Our facility provides researchers with access to four shRNA (human and mouse pGIPZ and pLKO) libraries housed at Rady Faculty of Health Sciences. Each individual bacterial clone contains a specific shRNA sequence predicted for a target human or mouse gene. Researchers can use these pre-cloned lentiviral shRNA clones for gene-specific silencing studies.

  • GIPZ Lentiviral shRNAmir Libraries (releases 1-13) from Open Biosystems

    • CMV promoter for efficient shRNAmir expression
    • Hairpin embedded in mir-30 increases knockdown
    • Turbo GFP marks  shRNAmir  expression
    • Puromycin selectable marker for stable RNAi
    • Unique molecular barcodes enable pooling experiments
    • Lentiviral vector allows RNAi in primary and non-dividing cells

    Biomedical functionality resource

    • Human ORF collection releases one and two
    • Human GIPZ lentiviral shRNAmir library release (release one to 13)
    • Mouse GIPZ lentiviral shRNAmir library release (release one to 13)
    • Human TRC shRNA library
    • Mouse TRC shRNA library
  • pGIPZ lentiviral vector diagram.

What we will do

We will retrieve the bacterial clone for the shRNA clones you order from the existing libraries (human shRNA pGIPZ releases 1-13, mouse shRNA pGIPZ releases 1-13, human shRNA pLKO,and mouse shRNA pLKO). We will provide the users a bacterial plate containing each of the lentiviral shRNA clone(s) they have requested.

What you need to supply

Users are advised to use the online engines below to check for availability of clones at UoM. If help is needed, you can provide us the gene ID in the service request form. We will check whether they are available in our established shRNA libraries. You will need to isolate/purify plasmids from the bacterial clone in their laboratories for downstream transfection or lentiviral vector packaging.

Suggested websites for clone search

GIPZ Lentiviral shRNA GPP Web Portal (search by gene)

  • Vector element Utility
    U6 Human U6 (RNA polymerase III) promoter provides high-level of expression in the target cells
    shRNA Simple stem-loop shRNA for gene knockdown
    hPGK Human phosphoglycerate kinase promoter drives expression of he puromycin resistance gene
    PuroR Puromycin resistance permits antibiotic-selective pressure and propagation of stable integrants
    RSV/5' LTR RSV promoter/5' long terminal repeat promotes strong lentiviral transcription in the packaging cells in the absence of Tat
    3' SIN LTR 3' self-inactivating long terminal repeat for increased lentivirus safety
    Ψ (psi) Psi packaging sequence allows viral genome packaging using lentiviral packaging systems
    RRE Rev response element enhances titer by increasing packaging efficiency full-length viral genomes
  • pLKO lentiviral vector diagram.

Packaging of lentiviral preparations

Production of lentiviral preparations

  • Using plasmids provided by users
  • Using shRNA from UM's shRNA library
  • Diagram of the replication process of HIV-1.
  • Replication cycle of HIV-1

    Upon binding of the HIV-1 virus to its cellular receptors (CD4 and chemokine receptor). There are three points to make:

    1. Nuclear import without the need of mitosis (vs retrovirus)
    2. Integration and stable gene expression (vs adenovirus)
    3. Ability to disrupt production of replicatioin-competent viruses
  • Production of replication-incompetent lentiviral vectors

    Lorem ipsum dolor sit amet, consectetur adipiscing elit, sed do eiusmod tempor incididunt ut labore et dolore magna aliqua. Ut enim ad minim veniam, quis nostrud exercitation ullamco laboris nisi ut aliquip ex ea commodo consequat.

  • Diagram of roduction of replication of incompetent lentiviral vectors.

Turnaround time

Our standard turnaround time for preparing viral preparations is seven working days. This means that if the user provides us with the purified lentiviral plasmid of interest, we should be able to prepare the virus within this timeframe.

For example, if the virus is made on a Tuesday, it will be harvested on the following Friday. If the virus is made on a Friday, it will be harvested on the following Monday.

What we will do

  • Maintenance of stocks of 293T cells, packaging and envelope plasmids (second or third generation) for lentiviral vector productions
  • Production of lentiviral preparations using customized plasmids or shRNA clones (from the UM's shRNA libraries) provided by users
  • Concentration of the viral preparations, if needed
  • Determination of the viral titer of the preparations (if direct EGFP measurement is possible)
  • If not, we will provide an estimate using the EGFP vector preparation we prepare side-by-side.

What you need to supply

High quality purified plasmid preparations (e.g., Qiagen Midiprep) at high concentration (e.g., 1 mg/ml).

Special considerations

Please discuss with the Core if you have a specific downstream workflow of your lentiviral vector work. We can provide suggestions for you to consider and/or incorporate in your research plan.

Other services

The following services are available upon request

  • Plasmid isolation
  • Transformation
  • Commonly used control shRNA lentiviral vectors for gene silencing experiments
  • EGFP-expressing lentiviral vectors (Cppt2E) for trouble-shooting or optimization of your transduction protocol
  • Consultation and training

LV vs Crispr/cas9

LV knockdown is a well-established technology that can achieve incomplete gene silencing and may have off-target effects, but it is still useful and versatile for validating gene functions, especially in primary cells and hard-to-transfect cells.

Crispr/cas9 is an evolving technology that allows for complete gene editing at the genome level, with the possibility of off-target effects (although improved versions have minimized this). It is highly useful for studies that involve cell lines and ES cells for longer-term analyses or downstream applications. However, depending on the cell types used, it can be time-consuming and the transfection efficiency of the cell line is a critical factor to consider.

Determining viral titers

To reduce costs, we won't determine the viral titer of every preparation. Instead, we will determine the titer and quality of a parallel EGFP-reporter lentiviral vector. However, if users request a specific titer determination, an extra fee of $30 will be charged for the setup of cell culture used in the transduction and/or the service fee required in running flow cytometry.

The lentiviral core can guarantee:

The lentiviral core can guarantee that all the reagents and cells used are optimal for viral preparations. The established lentiviral vector plasmids from the core should have viral titers of at least 1x106 I.U. per ml before concentration. Additionally, if it is confirmed that the transduction works, the core will repeat the remaking of the virus preparation for free.

The lentiviral core cannot guarantee:

The lentiviral core cannot guarantee that lentiviral transduction will work efficiently in all cell types, or that a specific shRNA clone from the libraries will effectively silence the gene of interest. Additionally, we cannot guarantee that all lentiviral vectors, including those from external sources, will be packaged at the same efficiency and high titers. Lastly, the core cannot guarantee high levels of expression for all transgenes.

Examples of applications

Lentiviral transduction of primary cells

There are several applications that use different techniques for specific purposes. Here are some examples:

  1. Lentiviral transduction of primary cells: This technique uses lentiviruses to introduce genetic material into primary cells. This can be useful for studying gene function or developing new therapies.

  2. Gene silencing: This technique involves suppressing the expression of a specific gene of interest to understand its role in cellular processes or disease. This can be done through methods such as RNA interference or CRISPR/Cas9 gene editing.

  3. Over-expression of genes: This technique involves increasing the expression of a gene of interest in order to study its function or rescue a phenotype caused by a mutation. This can be done using full-length or mutant copies of the gene.

  4. Generation of novel stable cell lines: This technique involves creating cell lines that have specific mutations, wild-type copies of genes, or shRNAs (small hairpin RNAs) that can be used to study gene function or disease.

  5. Production of secreted cytokines: This technique involves creating stable cell lines that express a specific cytokine of interest. The culture supernatant collected from these cell lines can then be used as a cheap source of the cytokine for further research.

Overall, these applications can be used in various fields, such as health care, research, education, and industry, to gain a better understanding of gene function, disease mechanisms, and develop new therapies.

Testing out lentiviral transduction

We suggested the use of concentrated Cppt2E (EGFP-expressing lentiviral vector) virus in testing transduction efficiency and in the optimizations of your cell type of interest.  

The use of concentrated Cppt2E virus (in stock item) in a titration experiment will also allow users to evaluate whether your cell type of interest requires the use of concentrated or unconcentrated virus format in your transduction experiments.

Troubleshooting transduction work

Are you experiencing issues with your lentiviral vector transduction work? Here are some possible factors to consider:

Have you successfully cloned and transfected your gene of interest or shRNA into the lentiviral vector?

Is your transduction protocol optimized for the specific cell type you are working with?

Is the promoter being used suitable for efficient transgene expression in your cells?

Have you measured the expression of your transgene to confirm successful transduction?


Protocols for virus preparation, titration, and infection of both adherent and suspension cells.

Infection protocol

Infection of adherent cells

The day before infection, seed 5x104 cells per well in a 24-well plate.

On the day of infection, remove the medium from the well.

Add 250 μl of virus supernatant that has been appropriately diluted (refer to the ‘virus preparation’ protocol below).

Incubate for 2 hours.

Discard the supernatant and add 250 μl of fresh medium.

Analyze the infected cells after 72 hours.

If puromycin selection is required:

After 72 hours, remove the medium from the well and transfer it to a tube. Add 500 μl of fresh medium to the well.

Spin the tube at 1200 rpm for 5 minutes, and resuspend the pellet in 500 μl of medium containing puromycin. Return these 500 μl back to the well.

On the next day, remove the medium from the well that is undergoing puromycin selection, and add 500 μl of fresh medium.

Note that the concentration and timing of puromycin selection may vary depending on the specific cell type.

Infection of suspension cells


Change medium of suspension cells one day before the experiment to ensure log phase growth.


On the day of infection, collect cells and count them.

Aliquot 1x105 cells per capped centrifuge tube and spin at 4000 rpm for 2 minutes.

Remove the supernatant and add 250 μl of virus supernatant appropriately diluted (refer to the ‘virus preparation’ protocol). Mix by pipetting.

Incubate for 2 hours at 37°C in an incubator.

After 2 hours, add 1 ml of fresh medium.

Pellet cells as in step 2, and remove the supernatant.

Add 0.5 ml of fresh medium to each tube and transfer the cells to a 24-well plate.

Analyze the infected cells after 72 hours.

If puromycin selection is required:

After 72 hours, remove the medium from the well and transfer it to a tube. Add 500 μl of fresh medium to the wells. Spin the tube at 1200 rpm for 5 minutes and resuspend the pellet in 500 μl of medium containing puromycin. Put these 500 μl back to the well.

On the next day, remove the medium from the well that is undergoing puromycin selection and add 500 μl of fresh medium.

Alternatively, you can follow this protocol:

Do a cell count (0.1x106 cells is best for transduction).

Add cells to an Eppendorf tube.

Centrifuge cells at 2000 rpm for 5 minutes.

Remove supernatant and resuspend pellet with 250 μl of virus suspension.

Put the solution in a 24-well plate.

Centrifuge at 2000 rpm for 1 hour at 24°C.

Remove supernatant (place pipette tip at the corner of the well and draw up liquid very slowly).

Resuspend pellet in 0.5 ml of cell culture medium.

Incubate at 37°C with 5% CO2.

Note that the concentration and timing of puromycin selection may vary from cell to cell.

Virus preparation

To prepare the virus for infection, follow these steps:

  • Centrifuge the virus stock (either concentrated by ultrafuge or pooled supernatant) to remove debris and filter before use.

  • Dilute the virus stock to the appropriate concentration for titration.

  • Add polybrene stock (1mg/ml, filtered and kept at -20c) to each virus solution at a final concentration of 8 ug/ml.

  • Use 250 ul of the virus solution (virus with polybrene) to infect cells.

For example, if you want an MOI of 20 IU/cell and you have a virus with a titre of 1.62x108 IU/ml, you can calculate the amount of virus to use as follows:

  • Infect: (0.5x106 cells)(20 IU/cell) = 1.0x107 IU
  • So, (1.0x107 IU) / (1.62x108 IU/ml) = 61.7μl of concentrated virus (in a final volume of 250 ul)
  • To make 300μl of diluted virus (for MOI 20) = 74μl conc. virus + 2.4 μl polybrene + 223.6 μl culture media

Remember to always adjust the amount of virus and polybrene according to the MOI and the number of cells to be infected.

Virus Titration with 293T Cells

The day before titration, place 5 x 104 cells/well of a 24-well dish.

On the day of titration, dilute virus as follows:

  • Dilute virus 100x to get 10x virus (mix 10μl virus with 90μl media)
  • Dilute 10x virus 10x to get 1x virus (mix 40μl 10x virus with 360μl media)
  • Dilute 1x virus 10x to get 1/10x virus (mix 30μl 1x virus with 270μl media)

Transfer 300μl of the 1x virus into a fresh tube.

  • Add 2.4μl of polybrene to the 300μl of 1x and 1/10x dilutions (final [PB] = 8μg/mL). Mix well.

  • Remove media from the well. Add 250μl of the diluted virus + PB. For a mock, add 250μl of plain media to one well (don’t add PB to the mock well).

  • Incubate for 2 hours.

  • Remove media. Add 1mL of fresh media.

  • After 3 days (Tuesday to Friday or Friday to Monday), harvest the wells.

  • Run flow analysis to determine the percentage of transfected cells (TF'd cells). Use the data from the dilution (1X or 1/10X) which provided the lowest percentage of TF'd cells. For example, if the 1X dilution resulted in 85% TF'd cells and the 1/10X dilution resulted in 15% TF'd cells, use the 1/10X dilution.

  • Calculate the titre using the following equation:

Titre = (1x105 cells) x (percentage of TF'd cells) x (4) x (dilution factor)

For example, if the flow analysis of the 1/10X dilution showed 10% TF'd cells, the titre would be:

Titre = (1x105 cells) x (10% TF'd cells) x (4) x (1000)

Titre = 4x107 PFU/mL (don't worry about the units)

Please note: You can request shRNA plasmid clones from the Manitoba Centre for Prometics & Systems Biology.

Price list

Custom-made virus preparations

Lentiviral construct provided by users (unconcentrated)

Volume Price
5 ml $150

Lentiviral construct provided by users (concentrated)

Volume Price
0.3 ml $500
0.5 ml $800
1 ml $1,000

Control vectors for our lentiviral libraries

Volume Price
*0.2 ml **$220
0.5 ml **$450
1 ml **$700

*A bigger volume than before.

** A price increase that reflects a dramatic increase in FBS price this year.

In-stock viral preparations (concentrated)

EGFP-reporter virus (cppt2E)

Volume Price
0.3 ml $500

Control vectors for our lentiviral libraries

Volume Price
0.3 ml $500

Clones from our libraries

Item Cost  
Human shRNA pGIPZ releases 1-13 $50 each  
Mouse shRNA pGIPZ releases 1-13 $50 each  
Human shRNA pLKO $50 each  
Mouse shRNA pLKO $50 each  

As a set, related clones targeting the same target gene have an additional charge of $25 each


Item Cost
Consultation on lentiviral work $180/hour
Hands-on training


Workshops No charge
Consultation on CRISPR work No charge

Contact us

Lentiviral Core Facility
Apotex Centre
750 McDermot Avenue West
University of Manitoba (Bannatyne campus)
Winnipeg, MB R3E 0T5 Canada


204-789 3921