Services

We offer comprehensive services in tissue culture and lentiviral vector production for University of Manitoba investigators, as well as provincial and international collaborators.

Understanding how a protein function in complex biological processes will always be key to basic biomedical research and identification of novel drug targets in human diseases. Advances in Technologies in delivery, gene silencing and genome-editing are evolving. Replication-incompetent HIV-1 based lentiviral vectors have become widely popular in the manipulations and functional analyses of a gene of interest in cells. Key to the successful uses of lentiviral vectors in these applications is the ability to produce high quality and high-titre lentiviral particles. The latter demands skills and a well-maintained system that can be time-consuming and frustrating to both new and experienced users.

Thanks to the financial supports from Research Manitoba and University of Manitoba, we have established a service platform that supports consistent production of high quality lentiviral particles at a very low cost for Manitoba researchers. It expands the users’ base of the existing Faculty of Medicine funded Biomedical Functionality Resource (that has housed 4 lentiviral vector shRNA libraries and two human ORFeome collections) on campus.

Director: Dr. Sam K.P. Kung
Technician: Ms. Manli Zhang

On this page

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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.

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.

Turnaround time

In general, we should be able to prepare the viral preparations within 7 working days if the user provides us the purified lentiviral plasmid of interest.

For example: A virus made on Tuesday would be harvested on Friday and a virus made on Friday would be harvested the next Monday.

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

Lentiviral constructs

Production of lentiviral constructs

  • Using plasmids provided by users
  • Using shRNA from the UM's shRNA library

Future demands of the Lentiviral Core platform

  • In-house production and support
  • Cheaper than buying viruses from commercial company (for the existing clones).  Estimated to be 17x cheaper than ordering the custom-made viruses from a commercial vendor.
  • Remains the method of choice for studying gene function in primary cells, and hard-to-transfect cells/cell lines. There are applications that current Crispr/Cas9 technology is not feasible.

LV vs Crispr/cas9

LV knockdown: well-established technology, incomplete gene silencing, off-target effects possible, but still useful and versatile in validating gene functions, especially in primary cells and hard-to-transfect cells.

Crispr/cas9: evolving technology, complete gene editing at genome level, off-target effects possible (but minimized with improved versions), highly useful in studies that involve cell lines and ES cells for longer term analyses or downstream applications. Depending on cell types, can be time-consuming. Transfection efficiency of the cell line of interest is a critical factor.

Determining viral titers

To cut down cost and price, we will not determine viral titer of each preparation.  We will determine the titer and quality of an EGFP-reporter lentiviral vector that we prepare in parallel with the viral preparations requested by flow cytometry.  This titer is routinely ~106/ml (without concentration) and ~108/ml (with concentration).

If the user requests a specific titer determination of the viral preparation, we will use either flow cytometry or puromycin selection (depending on the lentiviral vector used) to determine the titer.  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:
  • All the reagents and cells we use are optimal for viral preparations.
  • The viral titers of the established lentiviral vector plasmids from the Core should be at least 1x106 I.U. per ml before concentration.
  • A repeat of the re-making of the virus preparation for free, if it is confirmed that the transduction works.
The lentiviral core cannot guarantee:
  • Lentiviral transduction MUST work in your cell type to achieve good efficiency.
  • One of the shRNA clones of the libraries MUST work in gene silencing of your gene of interest
  • We can package ALL lentiviral vectors (local and external) of all generations at the same efficiency and high titers.
  • All the transgene are expressed at high level

Examples of applications

Lentiviral transduction of primary cells

Specific gene silencing of the target genes of interest in functional studies, and/or confirmation of the knockout mice phenotype, and validation of the candidate gene/protein revealed by proteomic and microarray data.

Over-expression of full-length cDNA or mutant cDNA of the gene interest in functional studies, and gene rescue experiments.

Generation of novel stable cell lines that harbour mutations, WT cDNA or shRNA of the target gene of interest.

Generation of stable cell lines that express secreted cytokine of interest. The culture supernatant collected in the culture will be a crude but cheap source of the cytokine a laboratory routinely needs in research.

diagram of lentiviral transduction of primary cells.

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

  • Is your lentiviral vector expressing your gene of interest or shRNA properly? (clonings, transfection?)
  • Is your transduction protocol optimized for your cell type? (transduction efficiency?)
  • Is the promoter good for the transgene expression in  your cells?  Any readout for your transgene expression?

Working with Lentiviral Vectors

To avoid any possible contamination and maintain a clean laboratory environment, we also recommend following these standard safety practices:

  • Wear double gloves, face protection, and lab coat at all times
  • Perform work in a limited access area in a Biological Safety Cabinet Class II, and post a biohazardous warning sign
  • Minimize splashes or aerosols with careful pipetting
  • Do not use needles, blades, etc
  • Decontaminate work surfaces at least once a day and after any spill of viable material
  • Decontaminate all cultures, stocks, and other biological wastes before disposal using approved decontamination methods, such as treatment with bleach and autoclaving. Before autoclaving, the biological materials should be placed in a sealed, durable, leak-proof container for transport from the laboratory.

Please follow University of Manitoba Standard Operating Procedures (PDF) for details.

Safety notice

Lentiviral vector preparations are replication incompetent.

BSL2 facility and an approved Biosafety permit are required in the transduction of cells. An update of the biological inventory to include the use and storage of lentiviral vectors in the users' laboratory biosafety permit is required.

Exercise caution when used in in vivo injections.

Protocols

Infection protocol

Infection of adherent cells

  • Seed 5x104 cells per well (24 well plate) 1 day before infection
  • On the day of infection, remove medium. Add 250 ul of viruse supernatant of appropriate dilutions (see’ virus preparation’ below)
  • Incubate for 2 hr
  • Discard the supernatant. Add 250 ul fresh medium
  • Analyze infected cells after 72 hr
If applicable
  • After 72 hr, puromycin selection. Take out medium, and put into tube. Add 500μl fresh medium to the wells. Spin the tube at 1200rpm for 5 minutes. Resuspend pellet in 500μl medium containing puromycin. Put these 500μl back to the well.
  • On the next day get out the medium of the well which is undergoing puromycin election, add 500μl fresh medium.

Concentration and timing of puromycin selection may vary from cell to cell.

Infection of suspension cells

  • Change medium of the suspension cells 1 day before the expt. To ensure log phase growth.
  • On the day of infection, collect cells and count.
  • Aliquot 1x105 cells per capped centrifuge tube. Spin at 4000rpm for 2 min.
  • Remove supernatant, and add 250 ul virus of appropriate dilutions (see “virus preparation”). Mix by pipetting.
  • Incubate for 2 hr at 37c (incubator).
  • After 2hr, add 1 ml fresh medium.
  • Pellet cells as in step. Remove supernatant.
  • Add 0.5 ml of fresh medium to each tube, and transfer the cells to a 24- well plate.
  • Analyze after 72hr
If applicable

After 72 hr puromycin selection. Take out medium, and put into tube. Add 500μl fresh medium to the wells. Spin the tube at 1200rpm for 5 minutes. Resuspend pellet in 500μl medium containing puromycin. Put these 500μl back to the well.

On the next day get out the medium of the well which is undergoing puromycin election, add 500μl fresh medium.

Alternative protocol
  • Do a cell count (0.1x106 cells best for transduction)
  • Add cells to eppendorf tube
  • Centrifuge cells at 2000 rpm for 5 minutes
  • Remove supernatant & resuspend pellet with 250μl of virus suspension
  • Put solution in 24 well plate
  • Centrifuge at 2000 rpm for 1 hour (24oC)
  • Remove supernatant (place pipet tip at corner of well and draw up liquid very slowly)
  • Resuspend pellet in 0.5ml of cell culture medium.
  • Incubate at 37oC, 5 per cent CO2

Concentration and timing of puromycin selection may vary from cell-to-cell.

Virus preparation

  • virus stock ( concentrated by ultrafuge or pooled supernatant ) must be centrifuged to remove debris and filtered before use.
  • Dilute virus stock to appropriate concentration for titration.
  • Add polybrene stock* 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.

*polybrene stock: 1mg/ml, filtered and kept at -20c.

EX. Cppt2E (40 per cent)

Titre = 1.62x108 IU/ml

Want MOI of 20 IU/cell

Infect: (0.5x106 cells)(20 IU/cells) = 1.0x107 IU

So (1.0x107 IU) / (1.62x108 IU/ml) = 61.7μl of conc. virus (in a final volume of 250 ul)

So to make 300μl of diluted virus (for MOI 20) = 74μl conc. virus + 2.4 μl polybrene + 223.6 μl culture media

Virus Titration with 293T Cells

1. Place 5 x 104 cells/well of a 24-well dish the day before titration.

2. On day of titration dilute virus (keep virus on ice as much as possible):

  • 100x à 10x (10μl virus in 90μl media)
  • 10x à 1x (40μl 10x in 360μl media)*
  • 1x à 1/10x (30μl 1x in 270μl media)
  • *Transfer 300μl of 1x into a fresh tube

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

4. Remove media from well. Add 250μl diluted virus + PB. Remember to add 250μl of plain media to one well for a mock (Don’t add PB to the mock well).

5. Incubate for 2 hours

6. Remove media. Add 1 mL fresh media.

7. After 3 days (Tues à Fri or Fri à Mon) harvest wells

8. Run flow to determine per cent of transfected cells (TF’d cells). Use the data from the dilution (1X or 1/10X) which provided the lowest per cent of Tf’d cells.

Example:

1X:  85 per cent Tf’d cells

Use this à  1/10X:  15 per cent Tf’d cells

9. Calculate the tire based on this equation:

Titre = (1x105 cells) x (per cent of Tf’d cells) x (4) x (dilution factor)

Example:

From flow analysis of 1/10X dilution we found 10 per cent Tf’d cells. What is the titre?

Titre = (1x105 cells) x (per cent of 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

Consultations

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

$180/hour
Workshops No charge
Consultation on CRISPR work No charge

Workshop

Past workshops

Contact us

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

 

204-789 3921