Introduction

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.

 

Materials

Types of Kits  

Product Catalog no.
BLOCK-iT™ Inducible H1 Lentiviral RNAi SystemK4925-00
BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector KitV488-20


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

Component
 
Catalog no.
 
V488-20K4925-00
pLenti4/BLOCK-iT™-DEST Gateway® Vector Kit
XX
Gateway® LR Clonase™ II Enzyme Mix
 X
One Shot® Stbl3™ Chemically Competent E. coliXX
pLenti6/TR Vector X
Blasticidin X
ViraPower™ Zeo Lentiviral Support Kit X
293FT Cell Line X
BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit X


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.

Box
Component
Shipping
Storage
1
pLenti4/BLOCK-iT™-DEST Gateway® Vector Kit
Room temperature
 -20°
 
2
Gateway® LR Clonase™ II Enzyme Mix
Dry ice
-20° C
3
One Shot® Stbl3™ Chemically Competent E. coli
Dry ice
-80° C
4pLenti6/TR VectorRoom temperature-20° C
5BlasticidinRoom temperature-20° C
6ViraPower™ Zeo Lentiviral Support KitBlue iceViraPower™ Packaging Mix: -20° C
Zeocin™: -20° C, protected from light
Lipofectamine® 2000: +4°  C (do not freeze)
7293FT Cell LineDry iceLiquid nitrogen
8-9BLOCK-iT™ Inducible H1 RNAi Entry Vector KitDry iceInducible H1 RNAi Entry Vector Reagents: -20° C
Tetracycline: -20° C, protected from light
One Shot® TOP10 Chemically Competent E. coli: -80° C


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.

Reagent
Composition
Amount
Lyophilized in TE Buffer, pH 8.0
6 µg
pLenti4-GW/H1/TO-lamin shRNA Control Plasmid
Lyophilized in TE Buffer, pH 8.0
10 µl
pLenti6/TR
Lyophilized in TE Buffer, pH 8.0
20 µg


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.

Reagent
Composition
Amount
Gateway® LR Clonase™ II Enzyme MixProprietary40 ml
Proteinase K Solution
2 µg/µl in:
10 mM Tris-HCl, pH 7.5
20 mM CaCl2
50% glycerol
 
40 ml
pENTR™-gus Positive Control
50 ng/µl in TE Buffer, pH 8.0
20 µl


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.

Reagent
Composition
Amount
S.O.C. Medium
2% Tryptone
0.5% Yeast Extract
10 mM NaCl
2.5 mM KCl
10 mM MgCl2
10 mM MgSO4
20 mM glucose
6 ml
Stbl3 Cells
--
21 x 50 µl
pUC19 Control DNA
10 pg/µl in 5 mM Tris-HCl, 0.5 mM EDTA, pH 8
50 µl


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.

Reagent
Composition
Amount
ViraPower Packaging Mix
Contains a mixture of the pLP1, pLP2, and pLP/VSVG plasmids, lyophilized in TE Buffer, pH 8.0
195 µg
Lipofectamine® 2000 Reagent
Proprietary
0.75 ml
Zeocin
100 mg/ml in sterile, deionized water
125 mg
Blasticidin
Powder
50 mg


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 10frozen 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
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Ordering Information

Catalog # Name Size List Price (USD) Qty
K492000 BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit 20 reactions 956.00
K492500 BLOCK-iT™ Inducible H1 Lentiviral RNAi System 20 reactions 2,260.00
V48820 BLOCK-iT™ Lentiviral RNAi Zeo Gateway® Vector Kit 20 reactions 1,294.00

Biosafety Features of the System

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).
 
For more information about the BL-2 guidelines and lentivirus handling, refer to the document, “Biosafety in Microbiological and Biomedical Laboratories”, 4th Edition, published by the Centers for Disease Control (CDC). This document may be downloaded from the Web at the following address:
 
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm
 
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.
 
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Experimental Outline

Generating an Entry Clone

 

Introduction

To express your shRNA of interest from pLenti4/BLOCK-iT™-DEST, you will first need to generate an entry clone in the pENTR™/H1/TO vector using the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit. General guidelines are provided below.
 
To express your shRNA of interest in a tetracycline-regulated manner, note that you must use the pENTR™/H1/TO entry vector to generate entry clones containing your shRNA sequence. Although a large selection of Gateway® entry vectors exists to facilitate generation of entry clones, only the pENTR™/H1/TO entry vector contains the elements required to facilitate proper regulated expression of shRNA molecules in mammalian cells. These elements include: 


  • The human H1/TO promoter, an RNA Polymerase III-dependent promoter that facilitates high-level, tetracycline-regulated expression of the shRNA of interest in mammalian cells (Hannon et al., 1991; Myslinksi et al., 2001).
  • A Polymerase III (Pol III) terminator for efficient transcription termination of the shRNA molecule. 

The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit is supplied with Catalog no. K4925-00, but is also available separately from Invitrogen.

Using pENTR™/H1/TO

To generate an entry clone in pENTR™/H1/TO, you will:

  • Design and synthesize two complementary oligonucleotides encoding your shRNA target sequence according to specified guidelines
  • Anneal the oligonucleotides to create a double-stranded oligonucleotide
  • Clone the double-stranded oligonucleotide into pENTR™/H1/TO using an optimized 5-minute ligation procedure
  • Transform competent E. coli and select for entry clones

For detailed instructions and guidelines to generate your entry clone, refer to the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit manual. This manual is supplied with Catalog no K4925-00, but is also available for downloading from our Web site (www.invitrogen.com) or by calling Technical Service.
Creating Expression Clones
 
After you have generated an entry clone, you are ready to perform the LR recombination reaction using your pENTR™/H1/TO entry construct and pLenti4/BLOCK-iT™-DEST vector to generate an expression clone. To ensure that you obtain the best possible results, we recommend that you read this section and the sections entitled Performing the LR Recombination Reaction and Transforming One Shot® Stbl3™ Competent E. coli before beginning.
 
Experimental Outline

To generate an expression clone, you will:
 
  1. Perform an LR recombination reaction using the attL-containing pENTR™/H1/TO entry clone and the attR-containing pLenti4/BLOCK-iT™-DEST vector. Note:  Both the entry clone and the destination vector should be supercoiled (see Important Note below).
  2. Transform the reaction mixture into a suitable E. coli host.
  3. Select for expression clones.

The pLenti4/BLOCK-iT™-DEST vector is supplied as a supercoiled plasmid. Although the Gateway® Technology manual has previously recommended using a linearized destination vector for more efficient LR recombination, further testing at Invitrogen has found that linearization of pLenti4/BLOCK-iT™-DEST is not required to obtain optimal results for any downstream application
 
Resuspending the Destination Vector

The pLenti4/BLOCK-iT™-DEST vector is supplied as 6 µg of plasmid, lyophilized in TE, pH 8.0. To use, simply resuspend the destination plasmid in 40 µl of sterile water to a final concentration of 150 ng/µl.
 
Propagating the Destination Vector

f you wish to propagate and maintain the pLenti4/BLOCK-iT™-DEST vector, we recommend using Library Efficiency® DB3.1™ Competent E. coli from Invitrogen (Catalog no. 11782-018) for transformation. The DB3.1™ E. coli strain is resistant to CcdB effects and can support the propagation of plasmids containing the ccdB gene.
 
Note:   Do not use general E. coli cloning strains including Stbl3™, TOP10, or DH5a for propagation and maintenance as these strains are sensitive to CcdB effects.
 
Guidelines to Propagate the Destination Vector

Follow the guidelines below when using DB3.1™ E. coli to propagate the pLenti4/BLOCK-iT™-DEST plasmid:

  • To maintain the integrity of the vector, select for transformants in media containing 50-100 µg/ml ampicillin and 15-30 µg/ml chloramphenicol.
  • Due to the potential for rearrangement of lentiviral vectors caused by recombination between the 5' and 3' LTRs (i.e. unwanted recombinants), we recommend analyzing transformants to verify the integrity of the destination vector before proceeding.
  • When propagating transformants, culture bacteria in LB media. Do not use “richer” bacterial media as these media tend to give rise to a greater number of unwanted recombinants.

Recombination Region of pLenti4/BLOCK-iT™-DEST

The recombination region of the expression clone resulting from pLenti4/BLOCK-iT™-DEST x pENTR™/H1/TO entry clone is shown below.
 
Features of the Recombination Region:

  • Shaded regions correspond to those DNA sequences transferred from the pENTR™/H1/TO entry clone into the pLenti4/BLOCK-iT™-DEST vector by recombination. Non-shaded regions are derived from the pLenti4/BLOCK-iT™-DEST vector.

Note:  The DNA sequences transferred from the pENTR™/H1/TO entry clone consist of an H1/TO RNAi cassette containing the human H1/TO promoter + your ds oligo encoding the shRNA of interest + Pol III terminator.

  • The transcriptional start site is indicated. Note that transcription starts at the first nucleotide following the end of the human H1/TO promoter sequence.
  • Bases 1868 and 3551 of the pLenti4/BLOCK-iT™-DEST sequence are:





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Performing the LR Recombination Reaction

Introduction

Follow the guidelines and instructions in this section to perform the LR recombination reaction using the pENTR™/H1/TO entry clone and the pLenti4/BLOCK-iT™-DEST vector. We recommend including a negative control (no LR Clonase™ II) in your experiment to help you evaluate your results.
 
Recommended E. coli Host

For optimal results, we recommend using Stbl3™ E. coli for transformation as this strain is particularly well-suited for use in cloning unstable DNA such as lentiviral DNA containing direct repeats.
One Shot® Stbl3™ Chemically Competent E. coli are included in the kit for transformation. For instructions, see Transforming One Shot® Stbl3™ Competent E. coli.   Note that transformants containing unwanted recombinants (see Note below) are obtained less frequently when Stbl3™ E. coli are used for transformation.

You may transform the LR recombination reaction into other recA, endA E. coli strains including TOP10 and DH5a™, if desired. Note however, that these strains are not as well-suited for cloning unstable DNA, and may give rise to a low percentage (< 5%) of transformants containing unwanted recombinants (i.e. plasmids where recombination has occurred between the 5' and 3' LTRs) when selected on plates containing only ampicillin. If you wish to use TOP10 or DH5a™ cells for transformation, follow the guidelines below to reduce the frequency of unwanted recombinants:


  • 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.

Do not transform the LR recombination reaction into E. coli strains that contain the F' episome (e.g. TOP10F'). These strains contain the ccdA gene and will prevent negative selection with the ccdB gene.
 
LR Clonase™ II Enzyme Mix

LR Clonase™ II enzyme mix is supplied with the kit (Catalog no. K4925-00 only) or available separately from Invitrogen to catalyze the LR recombination reaction. The LR Clonase™ II enzyme mix combines the proprietary enzyme formulation and 5X LR Clonase Reaction Buffer previously supplied as separate components in LR Clonase™ enzyme mix into an optimized single-tube format for easier set-up of the LR recombination reaction. Use the protocol provided on page 24 to perform the LR recombination reaction using LR Clonase™ II enzyme mix.

Note:   You may perform the LR recombination reaction using LR Clonase™ enzyme mix, if desired. To use LR Clonase™ enzyme mix, follow the protocol provided with the product. Do not use the protocol for LR Clonase™ II enzyme mix provided in this manual.

Positive Control for LR Reaction

The pENTR™-gus plasmid is included with the LR Clonase™ II enzyme mix for use as a positive control for the LR recombination reaction. You may use this entry clone in your LR recombination reaction to verify the efficiency of the LR reaction. However, the resulting expression clone cannot be used as an expression control because neither the pLenti4/BLOCK-iT™-DEST vector nor pENTR™-gus include a eukaryotic promoter to control expression of the gus gene in mammalian cells. For a map of pENTR™-gus, see the Appendix, page 90.

Materials Needed

You should have the following materials on hand before beginning:

  • 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

Setting Up the LR Recombination Reaction

Follow this procedure to perform the LR reaction between the pENTR™/H1/TO entry clone and the pLenti4/BLOCK-iT™-DEST vector. If you want to include a negative control, set up a separate reaction but omit the LR Clonase™ II enzyme mix.
 
  1.     Add the following components to 0.5 ml microcentrifuge tubes at room temperature and mix.

Component
Sample
Positive Control
Entry clone (50-150 ng/reaction)
1-7 µl
--
pENTR-gus (50 ng/µl)
--
2 µl
pLenti4/BLOCK-iT-DEST vector (150 ng/µl)
1 µl
1 µl
TE Buffer, pH 8.0
to 8 µl
5 µl

  2.     Remove the LR Clonase™ II enzyme mix from -20° C and thaw on ice (~ 2 minutes).
 
  3.     Vortex the LR Clonase™ II enzyme mix briefly twice (2 seconds each time).
 
  4.     To the sample above, add 2 µl of LR Clonase™ II enzyme mix. Mix well by pipetting up and down.
 
Reminder:   Return LR Clonase™ II enzyme mix to -20° C immediately after use.
 
  5.     Incubate the reaction at 25° C for 1 hour.
 
Note:   Extending the incubation time to 18 hours typically yields more colonies.
 
  6.     Add 1 µl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37° C.
 
  7.     Proceed to Transforming One Shot® Stbl3™ Competent E. coli

Note:  You may store the LR reaction at -20° C for up to 1 week before transformation, if desired.



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Transforming One Shot® Stbl3™ Competent E. coli

Introduction

Follow the instructions in this section to transform the LR recombination reaction into One Shot® Stbl3™ Chemically Competent E. coli (Box 8) included with the kit. The transformation efficiency of One Shot® Stbl3™ Chemically Competent E. coli is 1 x 108 cfu/µg plasmid DNA.
 
Materials Needed

You will need the following materials:

  • 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 

One Shot® Stbl3™ Transformation Procedure

Use this procedure to transform the LR recombination reaction into One Shot® Stbl3™ Chemically Competent E. coli.
 
  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.
 

What You Should See

When using One Shot® Stbl3™ Chemically Competent cells for transformation, the LR recombination reaction should result in greater than 4,000 colonies if the entire LR reaction is transformed and plated.
 
Confirming the Expression Clone

The ccdB gene mutates at a very low frequency, resulting in a very low number of false positives. True expression clones will be chloramphenicol-sensitive and ampicillin- and Blasticidin-resistant. Transformants containing a plasmid with a mutated ccdB gene will be chloramphenicol-, ampicillin-, and Blasticidin-resistant. To check your putative expression clone, test for growth on LB plates containing 30 µg/ml chloramphenicol. A true expression clone should not grow in the presence of chloramphenicol.
 
Sequencing

Sequencing the expression construct is not required as transfer of the H1/TO RNAi cassette from pENTR™/H1/TO into the pLenti4/BLOCK-iT™-DEST vector preserves the orientation of the cassette. However, if you wish to sequence the H1/TO RNAi cassette in pLenti4/BLOCK-iT™ -DEST, we recommend using the primers below.

Primer
Sequence
H1 Forward
5' -TGTTCTGGGAAATCACCATA-3'
V5(C-term) Reverse
5' -ACCGAGGAGAGGGTTAGGGAT-3'

Note:  For your convenience, Invitrogen has a custom primer synthesis service. For more information, see our web site (www.invitrogen.com) or call Technical Service.
 
Maintaining the Expression Clone

Once you have generated your expression clone, maintain and propagate the expression clone in LB medium containing 100 µg/ml ampicillin.

 
TOP

Producing Lentivirus in 293FT Cells

 

Introduction

Before you can create a stably transduced cell line expressing your miRNA, you will first need to produce a lentiviral stock (containing the packaged pLenti6/V5 expression construct) by co-transfecting the optimized ViraPower™ Packaging Mix and your pLenti6/V5-GW/miR expression construct into the 293FT Producer Cell Line. The following section provides protocols and instructions to generate a lentiviral stock.
 
Experimental Outline

To produce lentivirus in 293FT Cells, you will:
 
  1. Grow the 293FT Cells to obtain 6 x 106 293FT cells for each sample.

  2. Prepare plasmid DNA of your expression clone.

  3. Cotransfect the ViraPower™ Packaging Mix and pLenti6/V5-GW/miR expression plasmid DNA into 293FT Cells using Lipofectamine® 2000.

  4. Harvest virus-containing supernatants 48-72 hours post-transfection.
 
293FT Cell Line

The human 293FT Cell Line is supplied with the BLOCK-iT™ Lentiviral Pol II miR RNAi Kits to facilitate optimal lentivirus production (Naldini et al., 1996). The 293FT Cell Line, a derivative of the 293F Cell Line, stably and constitutively expresses the SV40 large T antigen from pCMVSPORT6TAg.neo and must be maintained in medium containing Geneticin®. For more information about pCMVSPORT6TAg.neo and how to culture and maintain 293FT cells, refer to the 293FT Cell Line manual. This manual is supplied with the BLOCK-iT™ Lentiviral Pol II miR RNAi Kits and is also available from our Web site (www.invitrogen.com) or by calling Technical Service  Make sure that cells are greater than 90% viable.
 
Note:   The 293FT Cell Line is available separately from Invitrogen.

The health of your 293FT cells at the time of transfection has a critical effect on the success of lentivirus production. Use of “unhealthy” cells can negatively affect the transfection efficiency, resulting in production of a low titer lentiviral stock. For optimal lentivirus production (i.e. producing lentiviral stocks with the expected titers), follow the guidelines below to culture 293FT cells before use in transfection:

  • Subculture and maintain cells as recommended in the 293FT Cell Line manual. Do not allow cells to overgrow before passaging. You will need 6 x 106 293FT cells for each sample.
  • Use cells that have been subcultured for less than 20 passages.

ViraPower™ Packaging Mix


The pLP1, pLP2, pLP/VSVG plasmids are provided in an optimized mixture to facilitate viral packaging of your pLenti6/V5-GW/miR expression vector following cotransfection into 293FT producer cells.
 
The amount of the packaging mix (195 µg) and Lipofectamine® 2000 Reagent (0.75 ml) supplied in the BLOCK-iT™ Lentiviral Pol II miR RNAi Kits is sufficient to perform 20 cotransfections in 10 cm plates using the recommended protocol. To use the ViraPower™ Packaging Mix, resuspend the contents of one tube (195 µg) in 195 µl of sterile water to obtain a 1 µg/µl stock.
 
Note:   ViraPower™ Packaging Mix is available separately from Invitrogen or as part of the ViraPower™ Bsd Lentiviral Support Kit.
 
Plasmid Preparation

Once you have generated your expression clone, you must isolate plasmid DNA for transfection. Plasmid DNA for transfection into eukaryotic cells must be very clean and free from contamination with phenol and sodium chloride. Contaminants will kill the cells, and salt will interfere with lipid complexing, decreasing transfection efficiency. We recommend isolating plasmid DNA using the PureLink™ Plasmid Purification Kits or CsCl gradient centrifugation.
 
Resuspend the purified pLenti6/V5-GW/miR expression plasmid in sterile water or TE Buffer, pH 8.0 to a final concentration ranging from 0.1-3.0 µg/µl. You will need 3 µg of the expression plasmid for each transfection.
 
Important:   Do not use mini-prep plasmid DNA for transfection.
 
Lipofectamine® 2000

The Lipofectamine® 2000 reagent supplied with the BLOCK-iT™ Lentiviral Pol II miR RNAi Kits is a proprietary, cationic lipid-based formulation suitable for the transfection of nucleic acids into eukaryotic cells (Ciccarone et al., 1999). Using Lipofectamine® 2000 to transfect 293FT cells offers the following advantages:

  • Provides the highest transfection efficiency in 293FT cells
  • DNA-Lipofectamine® 2000 complexes can be added directly to cells in culture medium in the presence of serum
  • Removal of complexes or medium change or addition following transfection is not required, although complexes can be removed after 4-6 hours without loss of activity

Note:   Lipofectamine® 2000 is available separately from Invitrogen or as part of the ViraPower™ Bsd Lentiviral Support Kit.
 
To facilitate optimal formation of DNA-Lipofectamine® 2000 complexes, we recommend using Opti-MEM® I

miR Positive Control

You may generate a miR Positive Control using the reagents included in the kit as follows:

  • Generate the pcDNA™6.2-GW/+EmGFP-miR-lacZ expression control using the lacZ double-stranded oligo and pcDNA™6.2-GW/+EmGFP-miR expression vector included with the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit and as described in the expression vector manual.
  • Use the pcDNA™6.2-GW/+EmGFP-miR-lacZ expression control to generate the lentiviral construct with pLenti6-V5-DEST vector using the Rapid BP/LR recombination reaction as described in this manual.
  • Use the resulting lentiviral expression construct, pLenti6/V5-GW/+EmGFP-miR-lacZ, to generate a miR control lentiviral stock (lacZ targeting miRNA).
  • Once generated, the miR control lentivirus may be transduced into mammalian cell lines to express an miRNA targeted to the human lacZ gene, and may be used as a control for the RNAi response in these cell lines.

pLenti6/V5-GW/lacZ Positive Control


A pLenti6/V5-GW/lacZ positive control vector is included with the pLenti6/V5-DEST vector for use as an expression control in the ViraPower™ Lentiviral Expression System. The b-galactosidase is expressed as a C-terminally tagged fusion protein that may be easily detected by western blot or functional assay.
 
To propagate and maintain the control plasmid:
 
  1. Resuspend the vector in 10 µl of sterile water to prepare a 1 µg/µl stock solution.

  2. Use the stock solution to transform a recA, endA E. coli strain like Stbl3™, TOP10, DH5a™-T1R, or equivalent. Use 10 ng of plasmid for transformation.

  3. Select transformants on LB agar plates containing 100 µg/ml ampicillin (for Stbl3™ cells) or LB agar plates containing 100 µg/ml ampicillin and 50 µg/ml Blasticidin (for TOP10 or DH5a).

  4. Prepare a glycerol stock of a transformant containing plasmid for long-term storage. Propagate the plasmid in LB containing 100 µg/ml ampicillin 

  5. Use the pLenti6/V5-GW/lacZ positive control to generate a control lentiviral stock (expressing the LacZ protein).

  6. Use the pLenti6/V5-GW/lacZ lentiviral control and the pLenti6/V5-GW/+EmGFP-miR-lacZ lentiviral control in cotransduction experiments as a positive control for lentiviral induced RNAi analysis in your system.


Materials Needed

You should have the following materials before starting:

  • pLenti6/V5-GW/miR expression construct (0.1-3.0 µg/µl in sterile water or TE Buffer, pH 8.0)
  • Positive controls (see above to generate positive controls; resuspend in sterile water to a concentration of 1 µg/µl)
  • ViraPower™ Packaging Mix (supplied with the kits; resuspend in 195 µl of sterile water to a concentration of 1 µg/µl)
  • 293FT cells cultured in the appropriate medium (i.e. D-MEM containing 10% FBS, 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids, and 1% penicillin/streptomycin). You will need 6 x 106 293FT cells for each sample.
  • Lipofectamine® 2000 transfection reagent (supplied with the kits; store at +4°C and mix gently before use)
  • Opti-MEM® I Reduced Serum Medium
  • Fetal bovine serum (FBS)
  • Complete growth medium containing sodium pyruvate (i.e. D-MEM containing 10% FBS, 2 mM L-glutamine, 0.1 mM MEM Non-Essential Amino Acids, 1% penicillin/streptomycin, and 1 mM MEM Sodium Pyruvate)   Note:   MEM Sodium Pyruvate provides an extra energy source for the cells and is available from Invitrogen as a 100 mM stock solution (Catalog no. 11360-070).
  • Sterile, 10 cm tissue culture plates (one each for lentiviral construct and controls)
  • Sterile, tissue culture supplies
  • 5 and 15 ml sterile, capped, conical tubes
  • Cryovials
 
Recommended Transfection Conditions

We produce lentiviral stocks in 293FT cells using the following optimized transfection conditions below. The amount of lentivirus produced using these recommended conditions at a titer of 1 x 105 to 1 x 107 transducing units (TU)/ml is generally sufficient to transduce 1 x 106 to 1 x 108cells at a multiplicity of infection (MOI) = 1.


Condition
Amount
Tissue culture plate size
10 cm (one per lentiviral construct)
Number of 293FT cells to transfect
6 x 106 cells
Amount of ViraPower Packaging Mix
9 µg (9 µl of 1 µg/µl stock)
Amount of pLenti6/V5-GW/miR expression plasmid
3 µg
Amount of Lipofectamine® 2000 Reagent to use
36 µl


Note:   You may produce lentiviral stocks using other tissue culture formats, but keep in mind that optimization will be necessary to obtain the expected titers.

TOP

Titering Your Lentiviral Stock

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.

  2. Example:  When using HT1080 cells, we usually plate 2 x 105 cells/well in a 6-well plate.

  3. 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.

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

  5. 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.

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

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

  8. 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.

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

  10. 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.

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

  12. 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.

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

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

  15. 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:

Lentivirus Dilution% EmGFP Positive Cell
10-291.5%
10-334.6%
10-44.4%


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).

TOP

Co-Transduction and Tetracycline-Regulated Expression

 

Introduction

Guidelines and instructions are provided in this section to co-transduce the Lenti6/TR and Lenti4/BLOCK-iT™-DEST lentiviral constructs into the mammalian cell line of choice, induce shRNA expression with tetracycline, and to assay for target gene knockdown. We recommend using this procedure if you have a single Lenti4/BLOCK-iT™-DEST lentiviral construct and you wish to verify that your shRNA of interest can be inducibly expressed in the mammalian cell line of interest.

If you have multiple Lenti4/BLOCK-iT™-DEST lentiviral constructs, we recommend first generating a stable cell line expressing the Tet repressor, and using this cell line as the host for your lentiviral constructs (see Generating a TetR-Expressing Host Cell Line).

Note:
   If you wish to constitutively express your gene of interest, simply transduce the Lenti4/BLOCK-iT™-DEST construct alone into cells at a suitable MOI.
 
When performing the co-transduction procedure, we highly recommend using titered Lenti6/TR and Lenti4/ BLOCK-iT™-DEST lentiviral stocks. Optimal expression results are generally obtained (i.e. low basal and high inducible target gene knockdown) when the Lenti6/TR construct is transduced into mammalian cells at a higher MOI than the Lenti4/BLOCK-iT™-DEST construct (see MOI to Use for Transduction). Depending on the cell line used and the nature of your target gene of interest, you may need to vary the ratio of Lenti6/TR lentivirus:Lenti4/BLOCK-iT™-DEST lentivirus transduced into host cells to optimize basal and induced shRNA expression levels. This is best accomplished when the titer of each lentiviral stock is known.
 
Experimental Outline

To express the shRNA of interest using the co-transduction procedure, you will:
 
  1. Transduce the Lenti6/TR lentiviral construct into mammalian cells at a suitable MOI (e.g. MOI = 10).

  2. Incubate cells for 24 hours, then transduce the Lenti6/TR-containing cells with the Lenti4/BLOCK-iT™-DEST lentiviral construct at a slightly lower MOI (e.g. MOI = 1-5).

  3. Incubate the cells for 24 hours, then remove the medium-containing virus.

  4. Incubate the cells for 24 hours, then add tetracycline to induce expression of the shRNA of interest. Alternatively, select for stably transduced cells using Blasticidin and Zeocin™, if desired. Once stable cell lines are generated, you may add tetracycline to induce expression of the shRNA of interest.
 
When performing the co-transduction procedure, you must transduce the Lenti6/TR lentiviral construct into mammalian cells before transducing the Lenti4/BLOCK-iT™-DEST expression construct to enable tetracycline-regulated expression of the shRNA of interest to occur. We generally wait at least 24 hours after transducing the Lenti6/TR construct before transducing the Lenti4/BLOCK-iT™-DEST construct to allow time for the Tet repressor to be expressed.

MOI to Use for Transduction

You may transduce the Lenti6/TR and Lenti4/BLOCK-iT™-DEST lentiviral constructs into your mammalian cell line at any suitable MOI (see Determining the Optimal MOI). Note however, that to sufficiently repress basal transcription of the shRNA of interest while still obtaining maximal levels of tetracycline-induced expression, we recommend transducing the Lenti6/TR construct into cells at a higher MOI than the Lenti4/BLOCK-iT™-DEST construct. As a starting point, we recommend transducing the Lenti6/TR construct into cells at an MOI of 10, and transducing the Lenti4/BLOCK-iT™-DEST construct into cells at an MOI of 1 to 5. You may optimize basal and tetracycline-induced expression levels by varying the MOI of the Lenti6/TR and/or Lenti4/BLOCK-iT™-DEST lentiviruses transduced.

Materials Needed

You should have the following materials on hand before beginning:

  • Titered Lenti6/TR lentiviral stock (store at -80°C until use)
  • Titered Lenti4/BLOCK-iT™-DEST lentiviral stock (store at -80° C until use)
  • Titered Lenti4-GW/H1/TO-laminshRNA lentiviral stock (if desired, 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 µg/ml tetracycline (supplied with Catalog no. K4925-00, Box 8; store protected from light)
  • 10 mg/ml Blasticidin stock (if selecting for stably transduced Lenti6/TR cells)
  • 100 mg/ml Zeocin™ stock (if selecting for stably transduced Lenti4/BLOCK-iT™-DEST cells)

Co-Transduction Procedure

Follow the procedure below to co-transduce the mammalian cell line of choice with the Lenti6/TR and your Lenti4/BLOCK-iT™-DEST lentiviral constructs to assay for tetracycline-regulated target gene knockdown. We recommend including a negative control (mock transduction) to help you evaluate your results. If you are selecting for stable cell lines, include two negative control samples, one for Blasticidin selection and the other for Zeocin™ selection.
 
  1. Plate cells in complete growth media as appropriate for your application.

  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. Twenty-four hours following transduction of Lenti6/TR virus (Day 2), thaw the Lenti4/BLOCK-iT™-DEST lentiviral stock and dilute (if necessary) the appropriate amount of virus (at a suitable MOI; recommended MOI = 1 to 5) into fresh complete medium. Keep the total volume of medium containing virus as low as possible to maximize transduction efficiency. DO NOT vortex.

  6. Remove the culture medium containing Lenti6/TR virus from the cells. Mix the medium containing Lenti4/BLOCK-iT™-DEST virus gently by pipetting and add to the Lenti6/TR virus-containing cells.

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

  8. Twenty-four hours following transduction of Lenti4/BLOCK-iT™-DEST virus (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 assay the cells 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: Remove the medium and replace with fresh, complete medium containing the appropriate amount of Blasticidin. Incubate the cells at 37° C for 24 hours, then trypsinize and replate cells into a larger-sized tissue culture format in fresh, complete medium containing Blasticidin and Zeocin™. Proceed to Step 9.

    Example:  If transducing cells in a 6-well format, trypsinize and replate cells into a 10 cm tissue culture plate before performing Blasticidin and Zeocin™ selection. For stable cell lines only

  9. 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).
     
    Note:   Transducing cells with Lenti6/TR and Lenti4/BLOCK-iT™-DEST lentivirus at a high MOI should result in most of the cells being Blasticidin- and Zeocin™-resistant. In this case, you may not be able to see distinct Blasticidin- and Zeocin™-resistant colonies when performing stable selection. You may also not see many non-transduced cells (i.e. dead cells).

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

  11. 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.


  12. 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 target gene knockdown from different Blasticidin- and Zeocin™-resistant clones. We recommend testing at least 10 Blasticidin- and Zeocin™-resistant clones and selecting the clone that provides the lowest basal and the highest level of induced target gene knockdown for further studies.
     

    Performing RNAi Analysis



    What You Should See



    Example of Expected Results

    Assaying for Lamin A/C Expression





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Generating a TetR-Expressing Host Cell Line

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).
  8.  
    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).

  9. 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.



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Examples of Expected Results

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).


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Troubleshooting

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.

Problem
Reason
Solution
Few or no colonies obtained from sample reaction and the transformation control gave colonies
Incorrect antibiotic used to select for transformants
Select for transformants on LB agar plates containing 100 µg/ml ampicillin.
 
LR recombination reaction not treated with proteinase K
Treat reaction with proteinase K before transformation.
 
Didn’t use the suggested amount of LR Clonase™ II enzyme mix or LR Clonase™ II enzyme mix was inactive
  • Make sure to store the LR Clonase™ II enzyme mix at -20° C.
  • Do not freeze/thaw the LR Clonase™ II enzyme mix more than 10 times.
  • Use the recommended amount of LR Clonase™ II enzyme mix
  • Test another aliquot of the LR Clonase™ II enzyme mix.
 Not enough LR reaction transformedTransform 2-3 µl of the LR reaction into One Shot® Stbl3™ Chemically Competent E. coli.
 Not enough transformation mixture platedIncrease the amount of E. coli plated.
 Did not perform the 1 hour grow-out period before plating the transformation mixtureAfter the heat-shock step, add S.O.C. Medium and incubate the transformation mixture for 1 hour at 37° C with shaking before plating.
 Too much entry clone DNA used in the LR reactionUse 50-150 ng of the entry clone in the LR reaction.
Different sized colonies (i.e. large and small) appear when using TOP10 E. coli for transformationSome transformants contain plasmids in which unwanted recombination has occurred between 5' and 3' LTRs
  • Select for transformants on LB plates containing both 100 µg/ml ampicillin and 50 µg/ml Zeocin™.
  • Use the One Shot® Stbl3™ Chemically Competent E. coli supplied with the kit for transformation. Stbl3™ E. coli are recommended for cloning unstable DNA including lentiviral DNA containing direct repeats and generally do not give rise to unwanted recombinants.
Few or no colonies obtained from the transformation controlCompetent cells stored incorrectly
  • Store the One Shot® Stbl3™ Chemically Competent E. coli at -80° C.
  • Thaw a vial of One Shot® cells on ice immediately before use.
 After addition of DNA, competent cells mixed by pipetting up and downAfter adding DNA, mix competent cells gently. Do not mix by pipetting up and down.




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.



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Problem
Reason
Solution
Low viral titer
Low transfection efficiency:
  • Used poor quality expression construct plasmid DNA (i.e. DNA from a mini-prep)
  • Unhealthy 293FT cells; cells exhibit low viability
  • Cells transfected in media containing antibiotics (i.e. Geneticin®)
  • Plasmid DNA:transfection reagent ratio incorrect
  • 293FT cells plated too sparsely
  • Do not use plasmid DNA from a mini-prep for transfection. Use the S.N.A.P.™ MidiPrep Kit or CsCl gradient centrifugation to prepare DNA. 
  • Use healthy 293FT cells under passage 20; do not overgrow.
  • Do not add antibiotics to media during transfection as this reduces transfection efficiency and causes cell death.
  • Use a DNA (in µg):Lipofectamine® 2000 (in µl) ratio ranging from 1:2 to 1:3.
  • Plate cells such that they are 90-95% confluent at the time of transfection OR use the recommended transfection protocol (i.e. add cells to media containing DNA:lipid complexes
 
Transfected cells not cultured in media containing sodium pyruvate
One day after transfection, remove media containing DNA:lipid complexes and replace with complete media containing sodium pyruvate. Sodium pyruvate provides an extra energy source for the cells.
 
Lipofectamine® 2000 Reagent handled incorrectly
  • Store at +4° C. Do not freeze.
  • Mix gently by inversion before use. Do not vortex
 Low viral titer, continued
Viral supernatant harvested too early
     
Viral supernatants can generally be collected 48-72 hours posttransfection. If many cells are still attached to the plate and look healthy at this point, wait an additional 24 hours before harvesting the viral supernatant.

 

Viral supernatant too dilute

Concentrate virus using any method of choice (Yee, 1999).
 

Viral supernatant frozen and thawed multiple times
Do not freeze/thaw viral supernatant more than 3 times.
 
Poor choice of titering cell line
Use HT1080 cells or another adherent cell line
 
Polybrene® not included during titering procedure
Transduce the lentiviral construct into cells in the presence of Polybrene®.
No colonies obtained upon titeringToo much antibiotic used for selectionDetermine the antibiotic sensitivity of your cell line by performing a kill curve experiment. Use the minimum amount of antibiotic required to kill your untransduced cell line.
 Viral stocks stored incorrectlyAliquot and store stocks at -80°C. Do not freeze/thaw more than 3 times.
 Polybrene® not included during transductionTransduce the lentiviral construct into cells in the presence of Polybrene®.
Titer indeterminable; cells confluentToo little antibiotic used for selectionIncrease amount of antibiotic used for selection.
 Viral supernatant not diluted sufficientlyTiter lentivirus using a wider range of 10-fold serial dilutions (e.g. 10-2 to 10-8).


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Transduction and Regulated shRNA Expression

The table below lists some potential problems and possible solutions that may help you troubleshoot your transduction and tetracycline-regulated knockdown experiment. Note that the troubleshooting tips provided in this section are based on the assumption that you are not constitutively expressing the shRNA of interest.

 

Problem
Reason                                           
Solution                                                                     
Low levels of gene knockdown observed after tetracycline induction                                                         
Low transduction efficiency
                                    
  • Polybrene® not included during transduction
  • Non-dividing cell type used
  • Transduce the lentiviral construct into cells in the presence of Polybrene®.
  • Transduce your lentiviral construct into cells using a higher MOI.
Low levels of gene knockdown observed after tetracycline induction, continuedMOI too low
Transduce your lentiviral construct into cells using a higher MOI.
 
Cells harvested and assayed too soon after addition of tetracycline
  • Do not assay for target gene knockdown until at least 48-72 hours after tetracycline addition to allow time for shRNA to be expressed and processed.
  • Place cells under Zeocin™ selection and generate a stable cell line prior to addition of tetracycline. Note: Placing cells under Zeocin™ selection can improve gene knockdown results by killing untransduced cells.
 
Target gene is important for cell viability
Place cells under Zeocin™ selection and generate a stable cell line prior to addition of tetracycline.
 
Viral stocks not titered

Titer the lentiviral stock before use.
 

Viral stock stored incorrectly
  • Aliquot and store stocks at -80° C.
  • Do not freeze/thaw more than 3 times
  • If stored for longer than 6 months, re-titer stock before use.
 
shRNA with weak activity chosenSelect a different target region. If possible, screen shRNA first by transient transfection of the pENTR™/H1/TO construct to verify its activity, then perform LR recombination with the pLenti4/BLOCK-iT™-DEST vector and proceed to generate lentivirus.
Note: Generally, transient transfection greatly overexpresses shRNA, so moderately active pENTR™/H1/TO entry clones may be less active when expressed from a lentiviral construct.
No tetracycline-regulated gene knockdown observed
Did not transduce the Lenti4/BLOCK-iT™-DEST lentiviral construct into a Tet repressor-expressing cell line
 
No tetracycline-regulated gene knockdown observed, continued
shRNA with no activity chosen                 
Select a different target region. If possible, screen shRNA first by transient transfection of the pENTR™/H1/TO construct to verify its activity, then perform LR recombination with the pLenti4/BLOCK-iT™-DEST vector and proceed to generate lentivirus.
     
 
Forgot to add tetracycline
After transducing the Lenti4/BLOCK-iT™-DEST lentivirus, add tetracycline to a final concentration of 1 µg/ml to induce expression of the shRNA of interest. Wait for at least 24 hours before assaying for target gene knockdown.
Cytotoxic effects observed after transductionTarget gene is essential for cell viabilityAfter transducing the Lenti4/BLOCK-iT™-DEST lentivirus, add Zeocin™ 



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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.

Problem
Reason
Solution                                            
Low levels of
tetracycline-regulated
gene knockdown
observed
Low transfection efficiency (if using Lipofectamine® 2000 Reagent)
  • Antibiotics added to the media during transfection
  • Cells too sparse at the time of transfection
  • Not enough plasmid DNA transfected
  • Not enough Lipofectamine® 2000 u

Do not add antibiotics to the media during transfection.

  • Plate cells such that they will be 90-95% confluent at the time of transfection.
  • Increase the amount of plasmid DNA transfected.
  • Optimize the transfection conditions for your cell line by varying the amount of Lipofectamine® 2000 used.
  • Select for a stable cell line.
 
Did not wait long enough after induction before assaying for gene knockdown
  • Repeat the transfection and wait for a longer period of time after induction before assaying for gene knockdown.
  • Perform a time course of expression to determine the point at which the highest degree of gene knockdown occurs.
 
ds oligo insert in your pENTR™/H1/TO construct contains mutations
When analyzing kanamycin-resistant transformants, sequence the ds oligo insert to verify its sequence. Select constructs containing the correct ds oligo insert for use in RNAi analysis.
 
shRNA sequence not optimal
due to:
  • Target region selected
  • Length of the shRNA sequence (i.e. stem length)
  • Loop sequence
  • Orientation of shRNA sequence
  • Verify that the shRNA sequence does not contain > 3 tandem T’s which can cause premature transcription termination.
  • Select a different target region.
  • Vary the length of the shRNA sequence (e.g. if the target sequence is 19 bp, try increasing the stem length 3 nucleotides)
  • Select a different loop sequence.
  • Vary the length of the loop.
  • Reverse the orientation of the shRNA hairpin sequence (e.g. change oligo sequence from sense-loop-antisense to antisense-loop-sense orientation).
 
Did not add enough tetracycline
Increase the amount of tetracycline used for induction
 
Targeted an essential gene
Generate a stable cell line, then add tetracycline to induce shRNA expression.
Gene knockdown observed, but not tetracycline-regulated
Did not transfect the pENTR™/H1/TO entry construct into a cell line expressing Tet repressor
Transfect the entry construct into a cell line that expresses Tet repressor:
  • Use one of Invitrogen’s T-REx™ Cell Lines
        OR
  • Generate your own stable TetR-expressing cell line using pcDNA™6/TR or pLenti6/TR, as desired.
Significant target gene knockdown observed in uninduced cells
Insufficient amount of Tet repressor expressed (when transfecting a stable TetR-expressing cell line)
Screen other TetR-expressing clones. Choose the clone that exhibit the highest level of TetR expression for use as the host for your pENTR™/H1/TO construct.
 
Co-transfected a TetR expression plasmid and the pENTR™/H1/TO construct
  • Use 6-fold more TetR expression plasmid
  • DNA than pENTR™/H1/TO plasmid DNA in the co-transfection.
  • Transfect the pENTR™/H1/TO construct into a cell line that stably expresses TetR.
 
When generating the TetR-expressing cell line, pcDNA™6/TR or pLenti6/TR construct introduced into a mammalian cell line in which the CMV promoter is down-regulated
Use a mammalian cell line in which the CMV promoter is not down-regulated as the host for the pcDNA™6/TR or pLenti6/TR construct.
No gene knockdown observed, even after tetracycline induction
shRNA with no activity chosen
  • Verify that the shRNA sequence does not contain > 3 tandem T’s which can cause premature transcription termination.
  • Select a different target region.
 
Hairpin designed incorrectly
Select the target sequence and design the singlestranded oligos.

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Vector Map 1- Map and Features of pLP1

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.
 

Feature
Benefit
Human cytomegalovirus (CMV) promoter
Permits high-level expression of the HIV-1 gag and pol genes in mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987).
Human ß-globin intron
Enhances expression of the gag and pol genes in mammalian cells.
HIV-1 gag coding sequence
Encodes the viral core proteins required for forming the structure of the lentivirus (Luciw, 1996).
HIV-1 pol coding sequence
Encodes the viral replication enzymes required for replication and integration of the lentivirus (Luciw, 1996).
HIV-1 Rev response element (RRE)
Permits Rev-dependent expression of the gag and pol genes
Human b-globin polyadenylation signal
Allows efficient transcription termination and polyadenylation of mRNA.
pUC origin of replication (ori)
Permits high-copy replication and maintenance in E. coli.
Ampicillin (bla) resistance gene
Allows selection of the plasmid in E. coli. 

Vector Map 2- Map and Features of pLP2

Features of pLP2

pLP2 (4180 bp) contains the following elements. All features have been functionally tested. 


Feature
Benefit
RSV enhancer/promoter
Permits high-level expression of the rev gene (Gorman et al., 1982).
HIV-1 Rev ORF
Encodes the Rev protein which interacts with the RRE on pLP1 to induce Gag and Pol expression, and on the pLenti4/BLOCK-iT-DEST expression vector to promote the nuclear export of the unspliced viral RNA for packaging into viral particles.
HIV-1 LTR polyadenylation signal
Allows efficient transcription termination and polyadenylation of mRNA.
Ampicillin (bla) resistance gene
Allows selection of the plasmid in E. coli.
pUC origin of replication (ori)
Permits high-copy replication and maintenance in E. coli.


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Vector Map 3 - Map of pENTR™-gus

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.

Vector Map 4 - Map of pLenti4-GW/H1/TO-lamin shRNA

Description

pLenti4-GW/H1/TO-laminshRNA is a 6656 bp control vector expressing an shRNA targeting the Lamin A/C gene. A double-stranded oligonucleotide encoding the lamin shRNA was cloned into the pENTR™/H1/TO vector using the reagents supplied in the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit to generate an entry construct containing the H1/TO-laminshRNA RNAi cassette. The H1/TO-laminshRNA RNAi cassette was transferred into the pLenti4/BLOCK-iT™-DEST vector using the Gateway® LR recombination reaction to generate the pLenti4-GW/H1/TO-laminshRNA vector. This vector has been fully sequenced.
 
Map of pLenti4-GW/H1/TO-laminshRNA
 
The complete sequence of pLenti4-GW/H1/TO-laminshRNA vector is available from our Web site (www.invitrogen.com) or by calling Technical Service.
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Recipes

LB (Luria-Bertani) Medium
1.0% Tryptone
0.5% Yeast Extract
1.0% NaCl
 pH 7.0
 
  1.     For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 10 g NaCl in 950 ml deionized water.
 
  2.     Adjust the pH of the solution to 7.0 with NaOH and bring the volume up to 1 liter.
 
  3.     Autoclave on liquid cycle for 20 minutes. Allow solution to cool to ~55°C and add antibiotic, if desired.
 
  4.     Store at +4°C.
 
LB Plates Containing Ampicillin and Blasticidin

Follow the instructions below to prepare LB agar plates containing ampicillin and Blasticidin.
 
Important  Note:   The stability of Blasticidin may be affected by high temperature, therefore, we do not recommend adding Blasticidin to warm LB agar. Let LB agar cool to room temperature before adding Blasticidin.
 
  1.    Prepare LB medium as above, but add 15 g/L agar before autoclaving.
 
  2.    Autoclave on liquid cycle for 20 minutes.
 
  3.    After autoclaving, cool to ~55°C, add ampicillin to a final concentration of 100 µg/ml and pour into 10 cm plates.
 
  4.    Let harden, then spread 50 µg/ml Blasticidin on each plate.
 
  5.    Invert and store at +4°C, in the dark. Plates containing Blasticidin may be stored at +4°C for up to 2 weeks.
 
Low Salt LB Medium with Zeocin™
10 g Tryptone
5 g Yeast Extract
5 g NaCl
 
  1.    Dissolve tryptone, yeast extract, and NaCl in 950 ml deionized water.
 
  2.    Adjust the pH of the solution to 7.5 with 5 M NaOH and bring the volume up to 1 liter. For plates, add 15 g/L agar before autoclaving.
 
  3.    Autoclave on liquid cycle for 20 minutes. Allow solution to cool to ~55°C and add Zeocin™ to a final concentration of 50 µg/ml.
 
  4.    Store at +4°C in the dark. Plates containing Zeocin™ are stable for 1-2 weeks.

Tetracycline
 
Use this procedure to prepare a 10 mg/ml stock solution from the tetracycline salt available separately from Invitrogen (Catalog no. Q100-19). Note that the tetracycline provided with the BLOCK-iT™ Inducible H1 Lentiviral RNAi System is supplied as a 10 mg/ml solution that is ready-to-use.
 
Important:   If you are using a different form of tetracycline (i.e. free base form), prepare the stock solution in 100% ethanol rather than water.
 
  1.    Weigh out 10 mg of tetracycline and transfer to a sterile microcentrifuge tube.
 
  2.    Resuspend the tetracycline in 1 ml of sterile water to produce a 10 mg/ml stock solution that is yellow in color.
 
  3.    Wrap the tube in foil and store the stock solution at -20°C, protected from exposure to light.
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Appendix -Zeocin™

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:

Organism
Zeocin Concentration and Selective Medium
E. coli
25-50 µg/ml in Low Salt LB medium*
Mammalian Cells
50-1000 µg/ml (varies with cell line)


*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.



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Appendix - Blasticidin

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.



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