Inducible shRNA Lentiviral Vectors

Introduction

The BLOCK-iT™ Inducible H1 Lentiviral RNAi System combines Invitrogen’s BLOCK-iT™ RNAi and ViraPower™ T-REx™ Lentiviral technologies to facilitate creation of a replication-incompetent lentivirus that delivers an inducible short hairpin RNA (shRNA) of interest to dividing or non-dividing mammalian cells for RNA interference (RNAi) analysis. The System includes:

• The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit for production of an entry clone that contains elements required for tetracycline-regulated expression of a double-stranded oligonucleotide (ds oligo) encoding an shRNA of interest in mammalian cells (i.e. human H1/TO promoter and RNA Polymerase III (Pol III) terminator). The entry vector containing this H1/TO RNAi cassette (H1/TO promoter + ds oligo + Pol III terminator) is used to transfer the H1/TO RNAi cassette into the lentiviral expression plasmid (see below) using Gateway® Technology.
• A promoterless pLenti4/BLOCK-iT™-DEST destination vector into which the H1/TO RNAi cassette of interest is transferred. This expression plasmid contains elements that allow packaging of the construct into virions and the Zeocin™ resistance marker for selection of stably transduced cell lines.
• The pLenti6/TR vector that constitutively expresses high levels of the tetracycline (Tet) repressor. This expression plasmid contains elements that allow packaging of the construct into virions and the Blasticidin resistance marker for selection of stably transduced cell lines.
• Components of the ViraPower™ T-REx™ Lentiviral System for production of a replication-incompetent lentivirus that transiently or stably expresses the shRNA of interest (after tetracycline induction) or Tet repressor in both dividing and non-dividing mammalian cells.

Advantages of the BLOCK-iT™ Inducible H1 Lentiviral RNAi System

Use of the BLOCK-iT™ Inducible H1 Lentiviral RNAi System to facilitate lentiviral-based, delivery of regulated shRNA to mammalian cells provides the following advantages:

• The pENTR™/H1/TO entry vector provides a rapid and efficient way to clone ds oligo duplexes encoding a desired shRNA target sequence into a vector containing an RNA Pol III-dependent, tetracycline-regulated expression cassette (i.e. H1/TO RNAi cassette) for use in RNAi analysis.
• The vectors in the System are Gateway®-adapted for easy transfer of the H1/TO RNAi cassette from the pENTR™/H1/TO vector into the pLenti4/BLOCK-iT™-DEST vector.
• Generates a replication-incompetent lentivirus that effectively transduces both dividing and non-dividing mammalian cells, thus broadening the potential RNAi applications beyond those of other traditional retroviral systems (Naldini, 1998).
• Efficiently delivers the shRNA of interest or the Tet repressor to mammalian cells in culture or in vivo. Expression of the shRNA of interest can be regulated by tetracycline.
• Includes a lentiviral construct expressing the Tet repressor to facilitate generation of a stable TetR-expressing cell line for rapid screening of multiple expression constructs.
• Provides stable, long-term expression of the shRNA of interest beyond that offered by traditional adenoviral-based systems.
• Produces a pseudotyped virus with a broadened host range (Yee, 1999).
• Includes multiple features designed to enhance the biosafety of the system.

The BLOCK-iT™ RNAi Technology

A large selection of BLOCK-iT™ RNAi products is available from Invitrogen to facilitate RNAi analysis in mammalian and invertebrate systems. The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit supplied with the BLOCK-iT™ Inducible H1 Lentiviral RNAi System uses a vector-based approach to allow efficient generation of H1/TO RNAi cassettes for regulated expression of shRNA molecules in mammalian cells. For constitutive expression of shRNA molecules in mammalian cells, use the BLOCK-iT™ U6 RNAi Entry Vector Kit or the BLOCK-iT™ Lentiviral RNAi Expression System. Other BLOCK-iT™ RNAi products are available to facilitate production and delivery of synthetic short interfering RNA (siRNA), diced siRNA (d-siRNA) or double-stranded RNA (dsRNA) for RNAi analysis in mammalian cells or invertebrate organisms, as appropriate. For more information about any of the BLOCK-iT™ RNAi products and other RNAi resources, see the RNAi Central application portal at www.lifetechnologies.com/RNAi

The ViraPower™ Lentiviral T-REx™ Technology

The ViraPower™ T-REx™ Lentiviral Technology combines Invitrogen’s ViraPower™ Lentiviral and T-REx™ technologies to facilitate highly efficient, tetracycline-regulated, in vitro or in vivo delivery of a target gene or RNA to dividing and non-dividing mammalian cells using a replication-incompetent lentivirus. Based on the lentikat™ system developed by Cell Genesys (Dull et al., 1998), the ViraPower™ T-REx™ Lentiviral Technology possesses features which enhance its biosafety while allowing high-level, regulated expression in a wider range of cell types than traditional retroviral systems. The main components of the ViraPower™ T-REx™ Lentiviral Technology include:

• A pLenti-based expression vector into which the DNA sequence of interest will be cloned. This vector contains elements required to allow packaging of the expression construct into virions and an antibiotic resistance marker to allow selection of stably transduced cell lines.
• The pLenti6/TR expression vector for high-level, constitutive expression of the Tet repressor under the control of a CMV promoter. This vector also contains elements to allow packaging of the construct into virions and the Blasticidin resistance marker for selection of stably transduced cell lines.
• The ViraPower™ Packaging Mix, an optimized mixture of the three packaging plasmids required for production of the lentivirus.
• An optimized 293FT cell line to facilitate optimal production of virus.

The Gateway® Technology

Gateway® Technology is a universal cloning method that takes advantage of the site-specific recombination properties of bacteriophage lambda (Landy, 1989) to provide a rapid and highly efficient way to move your DNA sequence of interest into multiple vector systems. To express your shRNA of interest in mammalian cells using the BLOCK-iT™ Inducible H1 Lentiviral RNAi System and Gateway® Technology, simply:

1. Clone a double-stranded oligonucleotide encoding your shRNA of interest into the pENTR™/H1/TO entry vector to create an entry clone. Transfect this entry clone directly into mammalian cells for initial screening, if desired.


2. Generate an expression clone by performing an LR recombination reaction between the pENTR™/H1/TO entry clone and the pLenti4/BLOCK-iT™-DEST vector.


3. Use your expression clone and the reagents supplied in the kit to produce lentivirus.


4. Transduce the lentiviral construct into TetR-expressing mammalian cells and add tetracycline to induce expression of the shRNA of interest. Select for stably transduced cells, if desired.

Types of Kits  

Kit Components
 
The BLOCK-iT™ Inducible H1 Lentiviral RNAi System and the BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector Kit include the following components.

Shipping/Storage

The BLOCK-iT™ Lentiviral RNAi Kits are shipped as described below. Upon receipt, store each item as detailed below. For more detailed information about the reagents supplied in the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit, refer to the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit manual.
 
Note:   The BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector Kit includes Boxes 1 and 3 only.

Vectors

The following vectors are included with the BLOCK-iT™ Inducible H1 Lentiviral RNAi System (Boxes 1 and 4). Store the vectors at -20°C.
 
Important: The BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector Kit does not include the pLenti6/TR vector.

Gateway® LR Clonase™ II Enzyme Mix

The following reagents are included with the Gateway® LR Clonase™ II Enzyme Mix (Box 2). Store at -20° C for up to 6 months. For long-term storage, store at -80°C.

Note: The pENTR™-gus control included with the LR Clonase™ II Enzyme Mix may be used as a positive control for the LR recombination reaction only . Do not use the resulting expression clone to produce lentivirus for expression purposes as the pLenti4/BLOCK-iT™-DEST vector does not contain a eukaryotic promoter and the gus gene will not be expressed in mammalian cells.
 
One Shot® Stbl3™ Chemically Competent E. coli

The following reagents are included with the One Shot® Stbl3™ Chemically Competent E. coli kit (Box 3). Transformation efficiency is US 1 x 10⁸ cfu/µg plasmid DNA. Store at -80° C.

ViraPower™ Lentiviral Support Kit Reagents

The table below lists the reagents included with the Blasticidin (Box 5) and ViraPower™ Zeo Lentiviral Support Kit (Box 6). Store as follows:

• ViraPower™ Packaging Mix and Blasticidin: -20° C
• Zeocin™: -20° C, protected from light
• Lipofectamine® 2000 Reagent: +4° C.

 
Important:  Do not freeze Lipofectamine® 2000 Reagent.

293FT Cell Line

The BLOCK-iT™ Inducible H1 Lentiviral RNAi System includes the 293FT Cell Line (Box 7) for production of lentiviral stocks. The 293FT Cell Line is supplied as one vial containing 3 x 10⁶  frozen cells in 1 ml of Freezing Medium. Upon receipt, store in liquid nitrogen.

 
BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit

The BLOCK-iT™ Inducible H1 Lentiviral RNAi System includes the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit to facilitate production of a Gateway® entry construct containing an H1/TO RNAi cassette for tetracycline-regulated expression of your short hairpin RNA (shRNA) of interest. The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit contains:

• Inducible H1 RNAi Entry Vector Reagents and Tetracycline (Box 8)
• One Shot® TOP10

Introduction

The lentiviral and packaging vectors supplied in the BLOCK-iT™ Inducible H1 Lentiviral RNAi System are third-generation vectors based on lentiviral vectors developed by Dull et al., 1998. This third-generation HIV-1-based lentiviral system includes a significant number of safety features designed to enhance its biosafety and to minimize its relation to the wild-type, human HIV-1 virus. These safety features are discussed below.
 
Biosafety Features of the BLOCK-iT™ Inducible H1 Lentiviral RNAi System

The BLOCK-iT™ Inducible H1 Lentiviral RNAi System includes the following key safety features:

• The pLenti4/BLOCK-iT™-DEST and pLenti6/TR vectors contain a deletion in the 3' LTR (DU3) that does not affect generation of the viral genome in the producer cell line, but results in “self-inactivation” of the lentivirus after transduction of the target cell (Yee et al., 1987; Yu et al., 1986; Zufferey et al., 1998). Once integrated into the transduced target cell, the lentiviral genome is no longer capable of producing packageable viral genome.
• The number of genes from HIV-1 that are used in the system has been reduced to three (i.e. gag, pol, and rev).
• The VSV-G gene from Vesicular Stomatitis Virus is used in place of the HIV-1 envelope (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994).
• Genes encoding the structural and other components required for packaging the viral genome are separated onto four plasmids (i.e. three packaging plasmid and pLenti4/BLOCK-iT™-DEST or pLenti6/TR). All four plasmids have been engineered not to contain any regions of homology with each other to prevent undesirable recombination events that could lead to the generation of a replication-competent virus (Dull et al., 1998).
• Although the three packaging plasmids allow expression in trans of proteins required to produce viral progeny (e.g. gal, pol, rev, env) in the 293FT producer cell line, none of them contain LTRs or the Y packaging sequence. This means that none of the HIV-1 structural genes are actually present in the packaged viral genome, and thus, are never expressed in the transduced target cell. No new replication-competent virus can be produced.
• The lentiviral particles produced in this system are replication-incompetent and only carry the gene of interest. No other viral species are produced.
• Expression of the gag and pol genes from pLP1 has been rendered Rev-dependent by virtue of the HIV-1 RRE in the gag/pol mRNA transcript. Addition of the RRE prevents gag and pol expression in the absence of Rev (Dull et al., 1998).
• A constitutive promoter (RSV promoter) has been placed upstream of the 5' LTR in the pLenti4/BLOCK-iT™-DEST and pLenti6/TR vectors to offset the requirement for Tat in the efficient production of viral RNA (Dull et al., 1998). 

Biosafety Level 2
 
Despite the inclusion of the safety features discussed above, the lentivirus produced with this System can still pose some biohazardous risk since it can transduce primary human cells. For this reason, we highly recommend that you treat lentiviral stocks generated using this System as Biosafety Level 2 (BL-2) organisms and strictly follow all published BL-2 guidelines with proper waste decontamination. Furthermore, exercise extra caution when creating lentivirus that express shRNA targeting human genes involved in controlling cell division (e.g. tumor suppressor genes).

Handle all lentiviruses in compliance with established institutional guidelines. Since safety requirements for use and handling of lentiviruses may vary at individual institutions, we recommend consulting the health and safety guidelines and/or officers at your institution prior to use of the BLOCK-iT™ Inducible H1 Lentiviral RNAi System.
 

• Select for transformants using Low Salt LB containing both 100 µg/ml ampicillin and 50 µg/ml Zeocin™. Note that transformed E. coli grow more slowly in LB media containing ampicillin and Zeocin™, and may require slightly longer incubation times to obtain visible colonies. For more information about Zeocin™, see the Appendix.
• Select small colonies for analysis as transformants containing a plasmid that has recombined between the 5' and 3' LTRs (i.e. unwanted recombinants) generally give rise to larger colonies than those containing an intact plasmid.

• Purified plasmid DNA of your pENTR™/H1/TO entry clone (50-150 ng/µl in TE Buffer, pH 8.0)
• pLenti4/BLOCK-iT™-DEST vector (150 ng/µl in TE Buffer, pH 8.0)
• pENTR™-gus control (if desired, supplied with Catalog no. K4925-00, Box 2)
• LR Clonase™ II enzyme mix (supplied with Catalog no. K4925-00, Box 2; store at -20° C until immediately before use)
• 2 µg/µl Proteinase K solution (supplied with Catalog no. K4925-00, Box 2; thaw and keep on ice until use)
• TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
• Sterile 0.5 ml microcentrifuge tubes

• LR recombination reaction•One Shot® Stbl3™ Chemically Competent E. coli (supplied with the kit, Box 8; one vial per transformation; thaw on ice immediately before use)
• S.O.C. Medium (supplied with the kit, Box 8; warm to room temperature)
• pUC19 positive control (if desired to verify the transformation efficiency; supplied with the kit, Box 8)
• LB Medium (if performing the pUC19 control transformation)
• 42 °C water bath
• LB plates containing 100 µg/ml ampicillin (two for each transformation; warm at 37 °C for 30 minutes before use)
• 37 °C shaking and non-shaking incubator 

1. Thaw, on ice, one vial of One Shot® Stbl3™ chemically competent cells for each transformation.


2. Add 2 to 3 µl of the LR recombination reaction into a vial of One Shot® Stbl3™ cells and mix gently. Do not mix by pipetting up and down. For the pUC19 control, add 10 pg (1 µl) of DNA into a separate vial of One Shot® cells and mix gently.


3. Incubate the vial(s) on ice for 30 minutes.


4. Heat-shock the cells for 45 seconds at 42 °C without shaking.


5. Remove the vial(s) from the 42 °C water bath and place them on ice for 2 minutes.


6. Add 250 µl of pre-warmed S.O.C. Medium to each vial.


7. Cap the vial(s) tightly and shake horizontally at 37 °C for 1 hour at 225 rpm in a shaking incubator.


8. Spread 25-100 µl of the transformation mix on a pre-warmed selective plate and incubate overnight at 37 °C. We recommend plating two different volumes to ensure that at least one plate will have well-spaced colonies. For the pUC19 control, dilute the transformation mix 1:10 into LB Medium and plate 25-100 µl.


9. Store the remaining transformation mix at +4 °C. Plate out additional cells the next day, if desired.

Introduction

Before proceeding to transduce the mammalian cell line of interest and express the miRNA for RNAi analysis, we highly recommend determining the titer of your lentiviral stock. While this procedure is not required for some applications, it is necessary if:

• You wish to control the number of integrated copies of the lentivirus
• You wish to generate reproducible gene knockdown results

 
Guidelines and protocols are provided in this section.
 
Titering Methods

You can determine the titer of your lentiviral stock using any of the following methods:

• Blasticidin selection (usually takes 2 weeks to determine the titer)
• EmGFP detection (usually takes 4 days post-transduction to determine the titer), if the miRNA sequence was cloned into pcDNA™6.2-GW/EmGFP-miR vector

Experimental Outline

To determine the titer of a lentiviral stock, you will:
 

1. Prepare 10-fold serial dilutions of your lentiviral stock.


2. Transduce the different dilutions of lentivirus into the mammalian cell line of choice in the presence of Polybrene®.


3. Based on the titering method used:

• Select for stably transduced cells using Blasticidin. Stain and count the number of Blasticidin-resistant colonies in each dilution.
• Determine the titer by flow cytometry 4 days post-transduction, if using EmGFP.

Factors Affecting Viral Titer

A number of factors can influence lentiviral titers including:

• The characteristics of the cell line used for titering.
• The age of your lentiviral stock. Viral titers may decrease with long-term storage at -80°C. If your lentiviral stock has been stored for longer than 6 months, we recommend titering or re-titering your lentiviral stock prior to use in an RNAi experiment.
• Number of freeze/thaw cycles. Viral titers can decrease as much as 10% with each freeze/thaw cycle.
• Improper storage of your lentiviral stock. Lentiviral stocks should be aliquotted and stored at -80°C.

Selecting a Cell Line

You may titer your lentiviral stock using any mammalian cell line of choice. Generally, we recommend using the same mammalian cell line to titer your lentiviral stock as you will use to perform your expression studies. However, in some instances, you may wish to use a different cell line to titer your lentivirus (e.g. if you are performing RNAi studies in a non-dividing cell line or a primary cell line). In these cases, we recommend that you choose a cell line with the following characteristics to titer your lentivirus:

• Grows as an adherent cell line
• Easy to handle
• Exhibits a doubling time in the range of 18-25 hours
• Non-migratory

We generally use the HT1080 human fibrosarcoma cell line (ATCC, Catalog no. CCL-121) for titering purposes.

Important:   You may use other cell lines including HeLa and NIH/3T3 to titer your lentivirus. However, note that the titer obtained when using HeLa cells or NIH/3T3 cells is approximately 10 fold lower than the titer obtained when using HT1080 cells.

• The titer of a lentiviral construct may vary depending on which cell line is chosen. If you have more than one lentiviral construct, we recommend that you titer all of the lentiviral constructs using the same mammalian cell line.

Blasticidin Selection

The pLenti6/V5-GW/miR expression construct contains the Blasticidin resistance gene (bsd) (Kimura et al., 1994) to allow for Blasticidin selection (Takeuchi et al., 1958; Yamaguchi et al., 1965) of mammalian cells that have stably transduced the lentiviral construct.

If you are using the BLOCK-iT™ Lentiviral Pol II miR RNAi Kits, Blasticidin is supplied with the kit. Blasticidin is also available separately from Invitrogen or as part of the ViraPower™ Bsd Lentiviral Support Kit (see page ix for ordering information). For more information about how to prepare and handle Blasticidin, and determine the Blasticidin sensitivity, refer to the Appendix.

Using Polybrene® During Transduction

Transduction of lentivirus into mammalian cells may be enhanced if cells are transduced in the presence of hexadimethrine bromide (Polybrene®). For best results, we recommend performing transduction in the presence of Polybrene®. Note, however, that some cells are sensitive to Polybrene® (e.g. primary neurons). Before performing any transduction experiments, you may want to test your cell line for sensitivity to Polybrene®. If your cells are sensitive to Polybrene® (e.g. exhibit toxicity or phenotypic changes), do not add Polybrene® during transduction. In this case, cells should still be successfully transduced.

Preparing and Storing Polybrene®

Follow the instructions below to prepare Polybrene® (Sigma, Catalog no. H9268):

1. Prepare a 6 mg/ml stock solution in deionized, sterile water.


2. Filter-sterilize and dispense 1 ml aliquots into sterile microcentrifuge tubes.


3. Store at -20°C for long-term storage. Stock solutions may be stored at -20°C for up to 1 year. Do not freeze/thaw the stock solution more than 3 times as this may result in loss of activity.  Note: The working stock may be stored at +4°C for up to 2 weeks.

Materials Needed

You will need the following materials:

• Your Lenti6/V5-DEST lentiviral stock (store at -80°C until use)
• Adherent mammalian cell line of choice
• Complete culture medium for your cell line
• 6 mg/ml Polybrene®, if desired
• 6-well tissue culture plates
• Blasticidin (10 mg/ml stock) and crystal violet (Sigma, Catalog no. C3886; prepare a 1% crystal violet solution in 10% ethanol), if you are using Blasticidin selection for titering
• Inverted fluorescence microscope and appropriate filters for EmGFP visualization, if you are using EmGFP titering method
• Phosphate-Buffered Saline (PBS; Invitrogen, Catalog no. 10010-023)

Preparing Mammalian Cells

Initiate your mammalian cell line of choice that will be used for titering. Grow the cells in the appropriate medium. You will use at least one 6-well plate for every lentiviral stock to be titered (one mock well plus five dilutions). Cells should be >95% viable.

Remember that you will be working with media containing infectious virus. Follow the recommended Federal and institutional guidelines for working with BL-2 organisms.

• Perform all manipulations within a certified biosafety cabinet.
• Treat media containing virus with bleach.
• Treat used pipets, pipette tips, and other tissue culture supplies with bleach and dispose of as biohazardous waste.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling viral stocks and media containing virus.

Transduction and Titering Procedure

Follow the procedure below to determine the titer of your lentiviral stock using the mammalian cell line of choice. Note:  If you have generated a lentiviral stock of the pLenti6-V5-GW/miR-lacZ control construct with or without EmGFP, perform titering using the Blasticidin or EmGFP method, and if you generated a lentiviral stock of the pLenti6-V5-GW/lacZ control construct, use Blasticidin titering method.

1. The day before transduction (Day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30-50% confluent at the time of transduction. Incubate cells at 37°C overnight.

Example:  When using HT1080 cells, we usually plate 2 x 10 ⁵ cells/well in a 6-well plate.


2. On the day of transduction (Day 2), thaw your lentiviral stock and prepare 10-fold serial dilutions ranging from 10-2 to 10-6. For each dilution, dilute the lentiviral construct into complete culture medium to a final volume of 1 ml. DO NOT vortex.
   
   Note:   You may prepare a wider range of serial dilutions (10-2 to 10-8), if desired.


3. Remove the culture medium from the cells. Mix each dilution gently by inversion and add to one well of cells (total volume = 1 ml). 


4. Add Polybrene® (if desired) to each well to a final concentration of 6 µg/ml. Swirl the plate gently to mix. Incubate at 37°C overnight.


5. The following day (Day 3), remove the media containing virus and replace with 2 ml of complete culture medium.


6. The following day (Day 4), proceed to Steps 7-8 for EmGFP titering method or proceed to Steps 9-14 for Blasticidin titering method.


7. Determine the titer by flow cytometry on Day 4 for titering EmGFP. For each viral dilution well of the 6 well plate, trypsinize and resuspend the cells in complete media at a concentration of 10-500 cells/ml.


8. Using a flow cytometry system, determine the percentage of GFP-positive cells for each dilution.


9. For Blasticidin selection, remove the medium on Day 4 and replace with complete culture medium containing the appropriate amount of Blasticidin to select for stably transduced cells.


10. Replace medium with fresh medium containing Blasticidin every 3-4 days.


11. After 10-12 days of selection (day 14-16), you should see no live cells in the mock well and discrete Blasticidin-resistant colonies in one or more of the dilution wells. Remove the medium and wash the cells twice with PBS.


12. Add crystal violet solution (1 ml for 6-well dish; 5 ml for 10 cm plate) and incubate for 10 minutes at room temperature.


13. Remove the crystal violet stain and wash the cells with PBS. Repeat wash.


14. Count the blue-stained colonies and determine your lentiviral stock titer.

Preparing Cells for Flow Cytometry

If you have used EmGFP titering method, prepare cells for flow cytometry according to the established protocols in use at your flow cytometry facility.  The steps below provide general guidelines, and other methods may be suitable.

1. At day 4 post-transduction, dissociate the cells from the plate by using trypsin or cell dissociation buffer.


2. Spin the cells at low speed to remove residual media components and resuspend the cell pellet in flow cytometry buffer such as calcium/magnesium free PBS with 1% FBS at the required density for analysis on your flow cytometer. Fixing the cells is not necessary for analysis, but may be done, if desired. Note: To fix your cells before flow cytometry, use 2% formaldehyde or paraformaldehyde in calcium/magnesium free PBS. However, these fixatives may increase autofluorescence of cells, thus it is critical to include fixed, mock-transduced cells as a negative control for flow cytometry.


3. Use the mock-transduced cells and the lowest dilution of virus (i.e. 10-2) as the negative and positive samples, respectively, to set up the parameters of your flow cytometer.

Calculating Lentiviral Titer

Calculate the EmGFP lentivirus titers from the dilutions at which the percentage of EmGFP-positive cells fall within the range of 1-30% (Sastry et al., 2002; White et al., 1999). This is to avoid analyzing dilution samples containing multiple integrated lentiviral genomes, which may result in an underestimate of the viral titer, or dilution samples containing too few transduced cells, which will give inaccurate results. Titer is expressed as transducing units (TU)/ml.

Use the following formula to calculate the titer: 

[F x C/V] x D

F = frequency of GFP-positive cells (percentage obtained divided by 100)

C = total number of cells in the well at the time of transduction

V = volume of inoculum in ml

D = lentivirus dilution

An example for calculating the lentiviral titer is provided below. An EmGFP lentiviral stock was generated using the above protocol. The following data were generated after performing flow cytometry analysis:

In the above example, the 10-4 dilution is used to calculate the titer since the percentage of EmGFP-positive cells falls into the desired range of 1-30%. The frequency of EmGFP-positive cells is 4.4/100 = 0.044, multiplied by 2 x 10⁵ (the number of cells in the well) divided by 1 (the volume of inoculum). Thus the calculation is as follows:

[(0.044 x 200,000)/1] x 10⁴

The lentiviral titer for this example is 8.8 x 10⁷ TU/ml.

What You Should See

When titering pLenti6/V5 lentiviral stocks using HT1080 cells, we generally obtain titers ranging from 5 x 10⁵ to 2 x 10⁷ transducing units (TU)/ml.

For an example of expected results obtained from a typical titering experiment using Blasticidin, see below.

Note: If the titer of your lentiviral stock is less than 1 x 10⁵ TU/ml, we recommend producing a new lentiviral stock. See the Troubleshooting section for more tips and guidelines to optimize your viral yield.

Example of Expected Results Using Blasticidin Titering Method

In this experiment, a pLenti6 lentiviral stock was generated using the previous protocol. HT1080 cells were transduced with 10-fold serial dilutions of the lentiviral supernatant (10-2 to 10-6 dilutions) or untransduced (mock) following the protocol. Forty-eight hours post-transduction, the cells were placed under Blasticidin selection (10 µg/ml). After 10 days of selection, the cells were stained with crystal violet (see plate below), and colonies were counted.

Thus, the titer of this lentiviral stock is 4.8 x 10⁶ TU/ml (i.e. average of 46 x 10⁵ and 5 x 10⁶).

Introduction

Once you have performed the co-transduction procedure and established that your Lenti4/BLOCK-iT™-DEST construct can be inducibly expressed, you may wish to establish a stable cell line that constitutively expresses the Tet repressor and inducibly expresses your shRNA of interest. We recommend first creating a stable cell line that expresses only the Tet repressor, then using that cell line as the host for your Lenti4/BLOCK-iT™-DEST lentiviral construct.
 
Several T-REx™ cell lines that stably express the Tet repressor are available from Invitrogen (see page x for ordering information). If you wish to assay for tetracycline-regulated expression of your gene of interest in 293, HeLa, CHO, or Jurkat cells, you may want to use one of the T-REx™ cell lines as the host for your Lenti4/BLOCK-iT™-DEST lentiviral construct.
 
Note:  The T-REx™ cell lines stably express the Tet repressor from the pcDNA™6/TR expression plasmid. This plasmid is used to generate stable TetR-expressing cell lines in Invitrogen’s T-REx™ System. Both pLenti6/TR and pcDNA™6/TR contain the same TetR gene. For more information about the T-REx™ cell lines or pcDNA™6/TR, see our Web site (www.invitrogen.com) or contact Technical Service.
 
Caution:   Although suitable for use in inducible gene expression experiments, the T-REx™-HeLa cell line has exhibited fairly significant basal level knockdown of the lamin A/C gene (~20%) after transduction with the Lenti4-GW/H1/TO-laminshRNA lentivirus. The level of Lamin A/C gene knockdown achieved after tetracycline addition is > 90%. If you are performing RNAi analysis of a gene involved in cellular growth control or viability, you may want to use another T-REx™ cell line or generate your own TetR-expressing cell line.

When generating a stable cell line expressing the Tet repressor, you will want to select for clones that express the highest levels of Tet repressor to use as hosts for your inducible Lenti4/BLOCK-iT™-DEST lentiviral construct. Those clones that express the highest levels of Tet repressor should exhibit the most complete repression of basal transcription of your shRNA of interest, resulting in the lowest levels of target gene knockdown in the absence of tetracycline.
 
Materials Needed

You should have the following materials on hand before beginning:

• Titered Lenti6/TR lentiviral stock (store at -80°C until use)
• Mammalian cell line of choice
• Complete culture medium for your cell line
• 6 mg/ml Polybrene®, if desired
• Appropriately sized tissue culture plates for your application
• 10 mg/ml Blasticidin stock solution

Lenti6/TR Transduction Procedure

Follow the procedure below to transduce the mammalian cell line of choice with the Lenti6/TR lentiviral construct and to use Blasticidin selection to generate a stable cell line. We recommend including a negative control (mock transduction) to help you evaluate your results.
 

1. Plate cells in complete growth media as appropriate.


2. On the day of transduction (Day 1), thaw the Lenti6/TR lentiviral stock and dilute (if necessary) the appropriate amount of virus (at a suitable MOI; recommended MOI = 10) into fresh complete medium. Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency. DO NOT vortex.


3. Remove the culture medium from the cells. Mix the medium containing virus gently by pipetting and add to the cells.


4. Add Polybrene® (if desired) to a final concentration of 6 µg/ml. Swirl the plate gently to mix. Incubate at 37°C overnight.


5. The following day (Day 2), remove the medium containing virus and replace with fresh, complete culture medium.


6. The following day (Day 3), remove the medium and replace with fresh, complete medium containing the appropriate amount of Blasticidin to select for stably transduced cells.


7. Replace medium with fresh medium containing Blasticidin every 2-3 days until Blasticidin-resistant colonies can be identified (generally 10-12 days after selection).
 
Note:   Transducing cells with Lenti6/TR lentivirus at a high MOI should result in most of the cells being Blasticidin-resistant. In this case, you may not be able to see distinct Blasticidin-resistant colonies when performing stable selection. You may also not see many non-transduced cells (i.e. dead cells).


8. Pick at least 10 Blasticidin-resistant colonies and expand each clone to assay for Tet repressor expression (see below). Alternatively, you may pool the heterogeneous population of Blasticidin-resistant cells and screen for Tet repressor expression.

 
Reminder: Integration of the lentivirus into the genome is random. Depending upon the influence of the surrounding genomic sequences at the integration site, you may see varying levels of Tet repressor expression from different Blasticidin-resistant clones. We recommend testing at least 10 Blasticidin-resistant clones and selecting the clone that provides the highest level of Tet repressor expression for use as the host for your Lenti4/BLOCK-iT™-DEST construct.
 
Detecting TetR Expression

To detect Tet repressor expression, we recommend performing Western blot analysis using an Anti-Tet repressor antibody (MoBiTec, Göttingen, Germany, Catalog. no. TET01).


Maintaining the TetR-Expressing Cell Line

Once you have generated your stable TetR-expressing cell line and have verified that the cells express suitable levels of Tet repressor, we recommend the following:

• Maintain your TetR-expressing cell line in medium containing Blasticidin
• Remember to freeze and store vials of early passage cells

 
Expressing the shRNA of Interest

To express the shRNA of interest in a tetracycline-regulated manner, use the TetR-expressing cell line as the host for your Lenti4/BLOCK-iT™-DEST lentiviral construct. After transduction, you have two options to express the shRNA of interest:

• You may add tetracycline and assay for transient target gene knockdown OR
• You may use Zeocin™ to select for a stable cell line, then add tetracycline to assay for target gene knockdown

Choose the option that best fits your needs.
 
Materials Needed

You should have the following materials on hand before beginning:

• Titered Lenti4/BLOCK-iT™-DEST lentiviral stock (store at -80°C until use)
• Your TetR-expressing host cell line cultured in medium containing Blasticidin
• Complete culture medium containing Blasticidin
• 6 mg/ml Polybrene®, if desired
• 10 mg/ml tetracycline (supplied with Catalog no. K4925-00, Box 8; store protected from light)
• Appropriately sized tissue culture plates for your application
• 100 mg/ml Zeocin™ stock (if selecting for stably transduced Lenti4/BLOCK-iT™-DEST cells)

Lenti4/BLOCK-iT™-DEST Transduction Procedure

Follow the procedure below to transduce your TetR-expressing cells with the Lenti4/BLOCK-iT™-DEST lentiviral construct and to use Zeocin™ to generate a stable cell line, if desired. We recommend including a negative control (mock transduction) to help you evaluate your results.
 

1. Plate the TetR-expressing cells in complete growth media as appropriate for your application. If you plan to select for stably transduced cells, plate cells such that they will be 50-60% confluent on the day of transduction.


2. On the day of transduction (Day 1), thaw the Lenti4/BLOCK-iT™-DEST lentiviral stock and dilute (if necessary) the appropriate amount of virus (at a suitable MOI; recommended MOI = 1-5) into fresh complete medium containing Blasticidin.Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency. DO NOT vortex.


3. Remove the culture medium from the cells. Mix the medium containing virus gently by pipetting and add to the cells.


4. Add Polybrene® (if desired) to a final concentration of 6 µg/ml. Swirl the plate gently to mix. Incubate at 37°C overnight.


5. The following day (Day 2), remove the medium containing virus and replace with fresh, complete medium containing Blasticidin. Incubate at 37° C overnight.


6. The following day (Day 3), perform one of the following:


• Transient knockdown experiments: Remove the medium containing virus and replace with fresh, complete medium containing 1 µg/ml tetracycline. Incubate the cells at 37° C for 24-48 hours before assaying for target gene knockdown. If you wish to perform the assay at a later time, continue to culture the cells or replate them into larger-sized tissue culture formats as necessary in medium containing tetracycline.
• Stable cell lines: Trypsinize and replate cells into a larger-sized tissue culture format in fresh, complete medium containing Blasticidin and Zeocin™. Proceed to Step 7.

Example:   If transducing cells in a 6-well format, trypsinize and replate cells into a 10 cm tissue culture plate in medium containing Blasticidin and Zeocin™.

For stable cell lines only



7. Replace medium with fresh medium containing Blasticidin and Zeocin™ every 2-3 days until Blasticidin- and Zeocin™-resistant colonies can be identified (generally 10-14 days after selection).


8. Pick at least 10 Blasticidin- and Zeocin™-resistant colonies and expand each clone. Alternatively, you may pool the heterogeneous population of Blasticidin- and Zeocin™-resistant cells.


9. Induce expression of the shRNA of interest by adding tetracycline to a final concentration of 1 µg/ml. Wait for the appropriate length of time (e.g. 24-48 hours) before assaying for target gene knockdown.




TOP

Example 1: Tetracycline-Regulated Knockdown of Lamin A/C in
T-REx™-HeLa Cells

In this experiment, double-stranded oligonucleotides targeting the endogenous lamin A/C gene and the lacZ reporter gene were generated and cloned into pENTR™/H1/TO using the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit. The H1/TO-lamin and H1/TO-lacZ RNAi cassettes were transferred into the pLenti4/BLOCK-iT™-DEST vector using the LR recombination reaction to generate the pLenti4-GW/H1/TO-laminshRNA and pLenti4-GW/H1/TO-lacZshRNA expression constructs. Lentiviral stocks were generated and titered in HT1080 cells following the protocols in this manual . 

T-REx™-HeLa cells plated in a 12-well plate were transduced with each lentiviral construct at an MOI of 3. Twenty-four hours after transduction, cells were trypsinized, replated, and placed under Zeocin™ (100 µg/ml) and Blasticidin (10 µg/ml) selection for 3 weeks. Zeocin™- and Blasticidin-resistant clonal isolates were plated and treated with 1 µg/ml tetracycline for 6 days. Cell lysates were prepared and equivalent amounts were analyzed by Western blot using an Anti-Lamin A/C Antibody (BD Biosciences, Catalog no. 612162) and an Anti-b-Actin Antibody (Abcam, Catalog no. ab6276).
 
Results:

• The lamin A/C-specific shRNA inhibits expression of the lamin A/C gene in a tetracycline-regulated manner, while no lamin A/C knockdown is observed with the lacZ-specific shRNA. The degree of lamin A/C knockdown observed is > 90% after tetracycline addition. Note: Some knockdown of Lamin A/C (~20%) is observed in the absence of tetracycline due to promoter leakiness.
• The degree of lamin A/C gene blocking achieved using the lamin shRNA is similar to that achieved with the well-characterized, chemically synthesized siRNA (Elbashir et al., 2001; Harborth et al., 2001).

Introduction

Review the information in this section to troubleshoot your recombination and lentiviral expression experiments.
 
LR Reaction and Transformation

The table below lists some potential problems and possible solutions that may help you troubleshoot the LR recombination and transformation procedures.

Generating the Lentiviral Stock

The table below lists some potential problems and possible solutions that may help you troubleshoot your co-transfection and titering experiments.

Transient Transfection and RNAi Analysis

The table below lists some potential problems and possible solutions that may help you troubleshoot your transient transfection and knockdown experiment.

pLP1 Map

Note that the gag and pol genes are initially expressed as a gag/pol fusion protein, which is then self-cleaved by the viral protease into individual Gag and Pol polyproteins. 
The complete sequence of pLP1 is available for downloading from our web site or by contacting Technical Service.
 
Features of pLP1

pLP1 (8889 bp) contains the following elements. All features have been functionally tested.
 

Description

pENTR™-gus is a 3841 bp entry clone containing the Arabidopsis thaliana gene for b-glucuronidase (gus) (Kertbundit et al., 1991). The gus gene was amplified using PCR primers containing attB recombination sites. The amplified PCR product was then used in a BP recombination reaction with pDONR201™ to generate the entry clone. For more information about the BP recombination reaction, refer to the Gateway® Technology with Clonase™ II manual which is available for downloading from our Web site or by contacting Technical Service.

Zeocin™ belongs to a family of structurally related bleomycin/phleomycin-type antibiotics isolated from Streptomyces. Antibiotics in this family are broad spectrum antibiotics that act as strong antibacterial and antitumor drugs. They show strong toxicity against bacteria, fungi (including yeast), plants, and mammalian cells (Baron et al., 1992; Drocourt et al., 1990; Mulsant et al., 1988; Perez et al., 1989).

The Zeocin™ resistance protein has been isolated and characterized (Calmels et al., 1991; Drocourt et al., 1990). This protein, the product of the Sh ble gene (Streptoalloteichus hindustanus bleomycin gene), is a 13.7 kDa protein that binds Zeocin™ and inhibits its DNA strand cleavage activity. Expression of this protein in eukaryotic and prokaryotic hosts confers resistance to Zeocin™.
 
Molecular Weight, Formula, and Structure

The formula for Zeocin™ is C60H89N21O21S3 and the molecular weight is 1,535. The diagram below shows the structure of Zeocin™.




Applications of Zeocin™

Zeocin™ is used for selection in mammalian cells (Mulsant et al., 1988); plants (Perez et al., 1989); yeast (Baron et al., 1992); and prokaryotes (Drocourt et al., 1990). Suggested concentrations of Zeocin™ for selection in mammalian cell lines and E. coli are listed below:

*Efficient selection requires that the concentration of NaCl be no more than 5 g/L (< 90 mM).

Handling Zeocin™

• High salt and acidity or basicity inactivate Zeocin™. Therefore, we recommend that you reduce the salt in bacterial medium and adjust the pH to 7.5 to keep the drug active. Note that the pH and salt concentration do not need to be adjusted when preparing tissue culture medium containing Zeocin™.
• Store Zeocin™ at -20°C and thaw on ice before use.
• Zeocin™ is light sensitive. Store the drug, and plates or medium containing drug, in the dark at +4°C. Culture medium containing Zeocin™ may be stored at +4°C protected from exposure to light for up to 1 month.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling Zeocin™-containing solutions.
• Zeocin™ is toxic. Do not ingest or inhale solutions containing the drug.

Preparing and Storing Zeocin™
Zeocin™ is supplied in autoclaved, deionized water in 1.25 ml aliquots at a concentration of 100 mg/ml. The stability of Zeocin™ is guaranteed for six months, if stored at -20°C protected from exposure to light.

Blasticidin S HCl is a nucleoside antibiotic isolated from Streptomyces griseochromogenes which inhibits protein synthesis in both prokaryotic and eukaryotic cells (Takeuchi et al., 1958; Yamaguchi et al., 1965). Resistance is conferred by expression of either one of two Blasticidin S deaminase genes: bsd from Aspergillus terreus (Kimura et al., 1994) or bsr from Bacillus cereus (Izumi et al., 1991). These deaminases convert Blasticidin S to a non-toxic deaminohydroxy derivative (Izumi et al., 1991).
 
Molecular Weight, Formula, and Structure

The formula for Blasticidin S is C17H26N8O5-HCl, and the molecular weight is 458.9. The diagram below shows the structure of Blasticidin.




Handling Blasticidin

Always wear gloves, mask, goggles, and protective clothing (e.g. a laboratory coat) when handling Blasticidin. Weigh out Blasticidin and prepare solutions in a hood.
 
Preparing and Storing Stock Solutions

Blasticidin may be obtained separately from Invitrogen (Catalog no. R210-01) in 50 mg aliquots. Blasticidin is soluble in water. Sterile water is generally used to prepare stock solutions of 5 to 10 mg/ml.

• Dissolve Blasticidin in sterile water and filter-sterilize the solution.
• Aliquot in small volumes suitable for one time use (see next to last point below) and freeze at -20°C for long-term storage or store at +4°C for short-term storage.
• Aqueous stock solutions are stable for 1-2 weeks at +4°C and 6-8 weeks at  -20°C.
• pH of the aqueous solution should be 7.0 to prevent inactivation of Blasticidin.
• Do not subject stock solutions to freeze/thaw cycles (do not store in a frost-free freezer).
• Upon thawing, use what you need and store the thawed stock solution at +4°C for up to 2 weeks.

Medium containing Blasticidin may be stored at +4°C for up to 2 weeks.