BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP

A MultiSite Gateway®-adapted, lentiviral destination vector for high-level knockdown by miRNA-based RNAi in dividing and non-dividing mammalian cells

The BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP (Emerald Green Fluorescent Protein) combines Invitrogen’s BLOCK-iT™ Pol II miR RNAi, ViraPower™ HiPerform™ Lentiviral, and MultiSite Gateway® technologies to facilitate the creation of a replication-incompetent lentivirus that delivers a microRNA (miRNA) sequence of interest to dividing or non-dividing mammalian cells for RNA interference (RNAi) analysis.

pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector

The pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector, included with the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP, carries the WPRE (Woodchuck Posttranscriptional Regulatory Element) from the woodchuck hepatitis virus, and cPPT (central polypurine tract) from the HIV-1 integrase gene. The pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector is designed to achieve elevated EmGFP expression, higher titers and higher expression of the knockdown cassette using a choice of promoters.


Components of the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP

The BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP includes the following components:

• The BLOCK-iT™ Pol II miR RNAi Expression Vector Kit with EmGFP. This vector kit is used for production of an expression clone containing a double-stranded oligonucleotide (ds oligo) encoding a pre-micro RNA (miRNA) sequence downstream of the EmGFP site for expression in mammalian cells using an RNA Polymerase II (Pol II) promoter, the human cytomegalovirus (CMV) immediate early promoter.
• The pDONR™221 vector is used as an intermediate to transfer the premiRNA expression cassette into the lentiviral expression plasmid using Gateway® Technology.
• A pLenti6.4/R4R2/V5-DEST destination vector into which the pre-miRNA cassette from the expression clone is transferred using Gateway® Technology. The pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector contains elements that allow packaging of the construct into virions, the WPRE and cPPT elements for higher titers relevant to knockdown and stronger EmGFP expression, and the Blasticidin resistance marker, which is driven by the murine PGK promoter for selection of stably transduced cell lines.
• The pENTR™5’/CMVp and pENTR™5’/EF-1αp entry vectors. Either of these vectors is introduced into the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector along with the miR RNAi cassette.(As an alternative, you can also use a Pol II promoter cloned using the pENTR™5′-TOPO® entry vector, sold separately.
• Gateway® BP Clonase™ II and Gateway® LR Clonase™ II Plus Enzyme Mixes are provided that facilitate the transfer of the pre-miRNA expression cassette from the expression vector along with the CMV, EF-1α, or other promoter into the pLenti6.4/R4R2/V5-DEST destination vector.
• The ViraPower™ Lentiviral Support Kit is provided for production of a replication-incompetent lentivirus that stably expresses the miRNA of interest in both dividing and non-dividing mammalian cells.

For additional information about the BLOCK-iT™ Pol II miR RNAi Technology, ViraPower™ HiPerform™ Lentiviral Technology, and Multisite Gateway® Technology,visit our web site at www.lifetechnologies.com, or contact Technical Support.

Advantages of the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP

Use of the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP to facilitate lentiviral-based delivery of miR RNAi to mammalian cells provides the following advantages:

• The BLOCK-iT™ Pol II miR RNAi Expression Vector with EmGFP provides a rapid and efficient way to clone ds oligo duplexes encoding a desired miRNA target sequence into a vector containing a Pol II promoter for use in RNAi analysis.
• Multisite Gateway®-adapted vectors allow easy transfer of the miR RNAi of interest from one expression vector (pcDNA™6.2-GW/EmGFP-miR) into another (pLenti6.4/R4R2/V5-DEST) along with the promoter of choice (CMV, EF-1α, or other).
• 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).
• The pLenti6.4 vectors included with this system contain the WPRE and cPPT elements to produce higher levels of EmGFP expression and higher functional titers than vectors that do not contain these elements.
• Efficiently delivers the miR RNAi of interest to mammalian cells in vitro or in vivo.
• Provides stable, long-term expression of the miR RNAi 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™ Pol II miR RNAi Technology

The BLOCK-iT™ Pol II miR RNAi Technology is a next generation RNAi technology employing miRNA expression vectors that allow flexible expression of miRNA-based knockdown cassettes driven by RNA Polymerase II (Pol II) promoters in mammalian cells. The BLOCK-iT™ Pol II miR RNAi Expression Vectors are specifically designed to allow expression of miRNA sequences and contain specific miR flanking sequences that allow proper processing  of the miRNA. The expression vector design is based on the miRNA vector system developed in the laboratory of David Turner (U.S. Patent Publication No. 2004/0053876) and includes the use of endogenous murine miR-155 flanking sequences. A variety of BLOCK-iT™ RNAi products are available from Invitrogen to facilitate RNAi analysis in mammalian and invertebrate systems. For more information about any of the BLOCK-iT™ RNAi products, see the RNAi Central application portal at www.lifetechnologies.com/rnai or contact Technical Support.

ViraPower™ HiPerform™ Lentiviral Technology

The ViraPower™ HiPerform™ Lentiviral Technology facilitates highly efficient in vitro 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™ HiPerform™ Lentiviral Technology possesses features which enhance its biosafety while allowing high-level expression in a wider range of cell types than traditional retroviral systems. The main components of the ViraPower™ HiPerform™ Lentiviral Expression System include:

• A pLenti-based expression vector (e.g. pLenti6.4/R4R2/V5-DEST) into which the DNA sequence of interest will b  cloned. This pLenti vector contains the WPRE and cPPT elements for higher levels of gene expression, with more cells expressing EmGFP and your miR RNAi cassette.. The vector also contains the elements required to allow packaging of the expression construct into virions (e.g., 5′ and 3′ LTRs, Ψ packaging signal).
• The ViraPower™ Packaging Mix which contains an optimized mixture of the three packaging plasmids, pLP1, pLP2, and pLP/VSVG. These plasmids supply the helper functions as well as structural and replication proteins in trans required to produce the lentivirus. For more information about the packaging plasmids, refer to the ViraPower™ HiPerform™ Lentiviral Expression System manual, which is available for downloading from www.lifetechnologies.com, or by contacting Technical Support.
• VSV Envelope Glycoprotein: Most retroviral vectors are limited in their usefulness as gene delivery vehicles by their restricted tropism and generally low titers. In the ViraPower™ HiPerform™ Lentiviral Expression System, this limitation has been overcome by use of the G glycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as a pseudotyping envelope, thus allowing production of a high titer lentiviral vector with a significantly broadened host cell range (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994).
• An optimized 293FT producer cell line that stably expresses the SV40 large T antigen under the control of the human CMV promoter and facilitates optimal production of virus. For more information about the 293FT Cell Line, refer to the 293FT Cell Line manual available for downloading from www.lifetechnologies.com, or by contacting Technical Support.

The MultiSite 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 transfer a single DNA sequence of interest into multiple vector systems. The MultiSite Gateway® Technology uses modifications of the Gateway® Technology to allow simultaneous cloning of multiple DNA fragments in a defined order and orientation to create an expression construct. In the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP, the MultiSite Gateway® Technology facilitates recombinational cloning of two DNA fragments encoding a promoter and miR RNAi of choice into the pLenti6.4/R4R2/V5-DEST lentiviral destination vector.
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To express your miR RNAi of interest in mammalian cells using the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP and Gateway® Technology:

1. Clone a double-stranded oligonucleotide encoding your miR RNAi sequence of interest into the pcDNA™6.2-GW/EmGFP-miR expression vector to create an expression clone.


2. Transfect this expression clone from Step 1 directly into mammalian cells for initial screening (if desired).


3. Transfer your pre-miRNA expression cassette into the pLenti6.4/R4R2/V5- DEST MultiSite Gateway® vector. To transfer your pre-miRNA expression cassette:
   • Generate an entry clone by performing a BP recombination reaction between the pcDNA™6.2-GW/EmGFP-miR expression clone and pDONR™221 donor vector, then proceed to step b.
   • Perform an LR recombination reaction between the resulting entry clone (pENTR™221/EmGFP-miR), a pENTR™5’ promoter construct, and pLenti6.4/R4R2/V5-DEST.




4. Use your lentiviral expression clone and the reagents supplied in the kit to produce a lentiviral construct.


5. Transduce the lentiviral construct into mammalian cells to express the miR RNAi.


6. Select for stably transduced cells, if desired.

For detailed information about the Gateway® Technology, refer to the Gateway® Technology with Clonase™ II manual which is available for downloading from our web site (www.lifetechnologies.com) or by contacting Technical Support.


Purpose of this Manual

This manual provides an overview of the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP and provides instructions and guidelines to:

1. Use the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector, pENTR™5’ promoter vector, and pcDNA™6.2-GW/EmGFP-miR expression clone in a Rapid BP/LR recombination reaction to generate a lentiviral expression clone containing the miR RNAi sequence of interest.


2. Co-transfect the pLenti6.4/Promoter/MSGW/EmGFP-miR expression construct and the ViraPower™ Packaging Mix into the 293FT cell line to produce a lentiviral stock.


3. Titer the lentiviral stock.


4. Transduce the lentiviral construct into mammalian cells and perform “transient” RNAi analysis.


5. Generate a stably transduced cell line, if desired.

For details and instructions to generate a pcDNA6.2™-GW/EmGFP-miR expression clone containing the miR RNAi expression cassette, refer to the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit manual. For instructions to culture and maintain the 293FT producer cell line, refer to the 293FT Cell Line manual. Both of these manuals are supplied with the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP and are also available for downloading from our web site (www.lifetechnologies.com) or by contacting Technical Support.

The One Shot® Stbl3™ Chemically Competent E. coli, BP Clonase™ II Enzyme Mix, LR Clonase™ II Plus Enzyme Mix, and Lipofectamine™ 2000 Reagent included in the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP are available separately from Invitrogen and are supplied with individual documentation detailing general use of the product. For instructions to use these products specifically with the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi System with EmGFP, follow the recommended protocols on this page.

Important:

The BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmFPG is designed to help you create a lentivirus to deliver and express a miR RNAi sequence in mammalian cells for RNAi analysis. Although this system is designed to help you express your miR RNAi sequence in the simplest, most direct fashion, use of the system is geared towards users that are familiar with the principles of retrovirus biology and gene silencing. We highly recommend that users possess a working knowledge of viral and tissue culture techniques, lipid-mediated transfection, Gateway® Technology, and the RNAi pathway. For more information about the following topics, refer to these published references:

• Retrovirus biology and the retroviral replication cycle: see Buchschacher and Wong-Staal, 2000 and Luciw, 1996.
• Retroviral and lentiviral vectors: see Naldini, 1999, Naldini, 1998, and Yee, 1999.
• RNAi pathway and expression of miR RNAi in mammalian cells: see published references (Brummelkamp et al., 2002; Cullen, 2004; Kim, 2005; McManus & Sharp, 2002; Sui et al., 2002; Yu et al., 2002; Zeng et al., 2002)

Introduction

RNA interference (RNAi) describes the phenomenon by which short, homologous RNA duplexes induce potent and specific inhibition of eukaryotic gene expression via the degradation of complementary messenger RNA (mRNA). RNAi is functionally similar to the processes of post-transcriptional gene silencing (PTGS) or cosuppression in plants (Cogoni et al., 1994; Napoli et al., 1990; Smith et al., 1990; van der Krol et al., 1990) and quelling in fungi (Cogoni & Macino, 1997; Cogoni & Macino, 1999; Romano & Macino, 1992). In plants, the PTGS response is thought to occur as a natural defense against viral infection or transposon insertion (Anandalakshmi et al., 1998; Jones et al., 1998; Li & Ding, 2001; Voinnet et al., 1999). In experimental settings, RNAi is widely used to silence genes through transfection of RNA duplexes or introduction of vector-expressed short hairpin RNA (shRNA).

The RNAi Pathway

In eukaryotic organisms, dsRNA produced in vivo, introduced by pathogens, or through research, is processed into 21-23 nucleotide double-stranded short interfering RNA duplexes (siRNA) by an enzyme called Dicer, a member of the RNase III family of double-stranded RNA-specific endonucleases (Bernstein et al., 2001; Ketting et al., 2001).

Each siRNA then incorporates into an RNA-induced silencing complex (RISC), an enzyme complex that serves to target cellular transcripts complementary to the siRNA for specific cleavage and degradation, or translational repression (Hammond et al., 2000; Nykanen et al., 2001). MicroRNAs (miRNAs) are endogenous RNAs that trigger gene silencing (Ambros, 2001; Carrington & Ambros, 2003).

miRNA Pathway

MicroRNAs (miRNAs) are endogenously expressed as small ssRNA sequences of ~22 nucleotides in length, which naturally direct gene silencing through components shared with the RNAi pathway (Bartel, 2004). Unlike shRNAs or siRNA, however, the miRNAs are found embedded, sometimes in clusters, in long primary transcripts (pri-miRNAs) of several kilobases in length containing a hairpin structure and driven by RNA Polymerase II (Lee et al., 2004), the polymerase also responsible for mRNA expression. Drosha, a nuclear RNase III, cleaves the stem-loop structure of the pri-miRNA to generate small hairpin precursor miRNAs (pre-miRNAs) which are ~70 nucleotides in length (Zeng et al., 2005). The pre-miRNAs are exported from the nucleus to the cytoplasm by exportin-5, a nuclear transport receptor (Lund et al.,2004; Yi et al., 2003). Following the nuclear export, the pre-miRNAs are processed by Dicer into a ~22 nucleotides miRNA (mature miRNA) molecule, and incorporated into an miRNA-containing RNA-induced silencing complex (miRISC) (Cullen, 2004).

Translational Repression versus Target Cleavage

The miR RNAi Vector

The BLOCK-iT™ Pol II miR RNAi Expression Vector System with EmGFP is supplied with the pcDNA™6.2-GW/EmGFP-miR vector that allows the expression of your engineered pre-miRNA. This vector allows expression of the engineered pre-miRNA under the control of the strong, Pol II human CMV (cytomegalovirus) promoter and Herpes Simplex virus (HSV) thymidine kinase (TK) polyadenylation signal. The coding sequence of EmGFP (Emerald Green Fluorescent Protein) is incorporated into the vector such that the pre-miRNA insertion site is in the 3’ untranslated (3’UTR) region of the fluorescent protein mRNA. Addition of EmGFP allows tracking of the miRNA expression and provides strong correlation of EmGFP expression with the knockdown of the target gene by your miRNA.

Advantages of Using Pol II miRNA-based Vector Systems

Using miRNA-based vector systems with Pol II promoters for RNAi cassette expression offer the following advantages over traditional siRNA or shRNA expression:

• Enables co-cistronic expression of reporter genes such as GFP (see above), allowing reliable tracking of miR RNAi expression in mammalian cells.
• Allows expression of miR RNAi from a variety of promoters, including tissue-specific and regulated promoters for in vivo experiments.
• Enables expression of multiple miR RNAi cassettes from a single transcript, allowing the knockdown of more than one gene simultaneously (see the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit manual for details).
• Permits design of predictable RNAi constructs with a high rate of success.

Human CMV Promoter

The BLOCK-iT™ Pol II miR RNAi Expression Vectors contain the human cytomegalovirus (CMV) immediate early promoter to allow high-level, constitutive miRNA expression in mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987). We have chosen the human CMV promoter to control vector-based expression of miR RNAi in mammalian cells for the following reasons:

• The promoter is recognized by RNA Polymerase II and controls high-level, constitutive expression of miRNA and co-cistronic reporter genes.
• The promoter is active in most mammalian cell types.

Note:   Although highly active in most mammalian cell lines, activity of the viral CMV promoter can be down-regulated in some cell lines due to methylation (Curradi et al., 2002), histone deacetylation (Rietveld et al., 2002), or both.


Structure of the Engineered premiRNA

The BLOCK-iT™ Pol II miR RNAi Expression Vectors are designed to accept engineered pre-miRNA sequences targeting your gene of interest. The engineered pre-miRNA sequence structure is based on the murine miR-155 sequence and the stem-loop structure was optimized to obtain a high knockdown rate. For details on miR-155 and stem-loop optimization, refer to the BLOCK-iT™ Pol II miR RNAi Expression Vector manual.
For optimized knockdown results, we recommend that the ds oligo encoding the engineered pre-miRNA have the following structural features:

• Two 4 nucleotides, 5’ overhangs complementary to the vector (required for directional cloning)
• A 5’G + short 21 nucleotide antisense sequence (mature miRNA) derived from the target gene, followed by
• A short spacer of 19 nucleotides to form the terminal loop and
• A short sense target sequence with 2 nucleotides removed (Δ2) to create an internal loop

The structural features are depicted below.
 
TGCT overhang    5' G + antisense       loop sequence     Sense Δ2 nt                 CAGG overhang
                             target sequence                                    target sequence


For more details on the structure and guidelines to design the oligonucleotides, refer to the BLOCK-iT™ Pol II mi  RNAi Expression Vector Kit manual.

Pre-miRNA Expression Cassette

The engineered pre-miRNA sequence is cloned into the cloning site of BLOCKiT ™ Pol II miR RNAi Expression Vectors that is flanked on either side with sequences from murine miR-155 to allow proper processing of the engineered pre-miRNA sequence. The pre-miRNA sequence and adjacent miR-155 flanking regions are denoted as the pre-miRNA expression cassette and are shown below. During the Gateway® recombination reactions, the pre-miRNA expression cassette is transferred between vectors.

EmGFP    5' miR flanking region     5' G + antisense    loop sequence     Sense Δ2 nt                3'miR flanking region
                                                      target sequence                                 target sequence

Once the engineered pre-miRNA expression cassette is introduced into the mammalian cells for expression, the pre-miRNA forms an intramolecular stemloop structure similar to the structure of endogenous pre-miRNA that is then processed by the endogenous Dicer enzyme into a 22 nucleotide mature miRNA. Note: The 21 nucleotides are derived from the target sequence while the 3’ most nucleotide is derived from the native miR-155 sequence.

Introduction

The BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP facilitates highly efficient, in vitro delivery of a miR RNAi sequence to dividing and non-dividing mammalian cells using a replication-incompetent
lentivirus.

Components of the System

The BLOCK-iT™ Pol II miR RNAi Expression Vector System with EmGFP contains:

• The pcDNA™6.2-GW/EmGFP-miR vector for production of an expression clone that contains elements required for expression of a double-stranded oligonucleotide encoding an miR RNAi sequence of interest in mammalian cells using a Pol II promoter. The expression vector containing the premiRNA expression cassette can be transfected into mammalian cells for transient RNAi analysis, or used to transfer the pre-miRNA expression cassette into the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector using Gateway® Technology. For detailed information about the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit and instructions to generate an expression clone, refer to the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit manual.

• The pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector allows easy transfer of the pre-miRNA expression cassette from the expression clone into a lentiviral destination vector for use with the Lentiviral system components. The destination vector contains the 5′ and 3′ LTRs, ψ packaging signals required to allow packaging of the expression construct into virions as well as a selectable marker to allow generation of stable cell lines.

• The pENTR™ 5’ - encoding a eukaryotic promoter of interest into a MultiSite Gateway® entry vector.

• The pDONR™221 vector is used as an intermediate to transfer the pre-miRNA expression cassette into the lentiviral expression plasmid using Gateway® Technology.

• Gateway® BP Clonase™ II and LR Clonase™ II Plus Enzyme Mixes allow the transfer of the pre-miRNA expression cassette from the expression vectors as well as a promoter of interest into the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector using the Rapid BP/LR recombination reaction.

• One Shot® Stbl3™ Competent E. coli to obtain optimal results with lentiviral DNA after transformation.

• ViraPower™ Packaging Mix that contains an optimized mixture of, pLP1, pLP2, and pLP/VSVG. These packaging plasmids supply helper functions as well as structural and replication proteins in trans to produce the lentivirus.

• VSV Envelope Glycoprotein: Most retroviral vectors are limited in their usefulness as delivery vehicles by their restricted tropism and generally low titers. In the BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP, this limitation is overcome by use of the G glycoprotein gene from Vesicular Stomatitis Virus (VSV-G) as a pseudotyping envelope, thus allowing production of a high titer lentivirus with a significantly broadened host cell range (Burns et al., 1993; Emi et al., 1991; Yee et al., 1994).

• An optimized 293FT producer cell line that stably expresses the SV40 large T antigen under the control of the human CMV promoter and facilitates optimal production of virus. For more information refer to the 293FT Cell Line manual.

System Overview

To use the The BLOCK-iT™ Pol II miR RNAi Expression Vector System with EmGFP, co-transfect the ViraPower™ Packaging Mix and the pLenti6.4 expression construct containing the pre-miRNA expression cassette into 293FT cells to produce a replication-incompetent lentivirus, which can then be transduced into the mammalian cell line of interest. Once the lentivirus enters the target cell, the viral RNA is reverse-transcribed, actively imported into the nucleus (Lewis & Emerman, 1994; Naldini, 1999), and stably integrated into the host genome (Buchschacher & Wong-Staal, 2000; Luciw, 1996). Once the lentiviral construct has integrated into the genome, the miR RNAi is constitutively expressed, allowing you to perform transient RNAi analysis or use Blasticidin selection to generate   stable cell line for long-term knockdown studies.


Features of the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector

The pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector contains the following elements:

• Rous Sarcoma Virus (RSV) enhancer/promoter for Tat-independent production of viral mRNA in the producer cell line (Dull et al., 1998)
• Modified HIV-1 5′ and 3′ Long Terminal Repeats (LTR) for viral packaging and reverse transcription of the viral mRNA (Dull et al., 1998; Luciw, 1996) Note: The U3 region of the 3′ LTR is deleted ( U3) and facilitates self-inactivation of the 5′ LTR after transduction to enhance the biosafety of the vector (Dull et al., 1998)
• HIV-1 psi (Ψ) packaging sequence for viral packaging (Luciw, 1996)
• HIV Rev response element (RRE) for Rev-dependent nuclear export of unspliced viral mRNA (Kjems et al., 1991; Malim et al., 1989)
• HIV-1 central polypurine tract (cPPT) for efficient import of the pro-virus into the nucleus of transduced cells (Park, 2001)
• Woodchuck hepatitis virus post-transcriptional regulatory element (WPRE) for high levels of expression of the viral genome during production and EmGFP after transduction and integration (Zufferey et al., 1998)
• Option of use of the human CMV promoter for high-level, constitutive expression of the miR RNAi, the non-viral human EF-1α promoter for lower but more ubiquitous expression in primary cells and in vivo with lower risk of promoter shut down, or your own promoter of interest cloned into the pENTR™5’/TOPO® vector (sold separately)
• Two recombination sites, attR4 and attR2, for recombinational cloning of the miR RNAi of interest from the pcDNA™6.2-GW/EmGFP-miR expression clone using MultiSite Gateway® Technology
• Chloramphenicol resistance gene (CmR) located between the two attR sites for counterselection
• The ccdB gene located between the attR sites for negative selection
• Blasticidin resistance gene (Izumi et al., 1991; Kimura et al., 1994; Takeuchi et al., 1958; Yamaguchi et al., 1965) for selection in E. coli and mammalian cells
• Ampicillin resistance gene for selection in E. coli
• pUC origin for high-copy replication of the plasmid in E. coli

• pENTR™ 5′-TOPO® containing the CMV promoter, EF-1α promoter entry clone, or your promoter of interest: attL4 and attR1

• pcDNA6.2-GW/EmGFP-miR expression clone containing EmGFP and your miR RNAi of interest: attB1 and attB2

• pDONR™221 vector for conversion of the miR RNAi expression clone into an attL1 and attL2 entry clone

• pLenti6.4/R4R2/V5-DEST lentiviral destination vector: attR4 and attR2

1. Generate an entry clone by performing a BP recombination reaction between the attB substrate (pcDNA™6.2-GW/EmGFP-miR expression clone) and attP substrate (pDONR™221 vector) using BP Clonase™ II Enzyme Mix.


2. Perform an LR recombination reaction between the resulting entry clone (attL substrate), a 5’ entry clone (att  substrate) carrying a promoter, and the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector (attR substrate) using LR Clonase™ II Plus Enzyme Mix. The standard BP and LR recombination reaction requires more than 2 days for completion. See below for details on expressing the miRNA from pLenti6.4/R4R2/V5-DEST destination vector using the Rapid BP/LR Recombination Reaction.

1. Perform a BP recombination reaction between the pcDNA™6.2-GW/EmGFPmiR expression clone and the pDONR™221 vector using BP Clonase™ II Enzyme Mix. This recombination reaction yields a pENTER™221/EmGFP-miR entry clone.


2. Perform an LR recombination reaction between the pENTR™221/EmGFP-miR entry clone (Step 1) the pENTR™5’ promoter clone, and pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector to produce a lentiviral expression clone.

• rrnB T1 and T2 transcription terminators for protection of the EmGFP gene and miR RNAi from expression by vector-encoded promoters
• Two recombination sites, attP1 and attP2, for recombinational cloning of the gene of interest from a Gateway® expression clone or attB PCR product
• ccdB gene located between the two attP sites for negative selection
• Chloramphenicol resistance gene located between the two attP sites for counterselection
• Kanamycin resistance gene for selection in E. coli
• pUC origin for replication and maintenance of the plasmid in E. coli

Introduction

The lentiviral and packaging vectors supplied in the BLOCK-iT™ HiPerform™ Lentiviral RNAi Expression System with EmGFP are third-generation vectors based on lentiviral vectors developed by Dull et al., 1998. This third-generation 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™ HiPerform™ Lentiviral RNAi Expression System

The BLOCK-iT™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP includes the following key safety features:

• The pLenti6.4/R4R2/V5-DEST expression vector contains a deletion in the 3′ LTR (ΔU3) 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. 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 Ψ 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 i  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 pLenti6.4/R4R2/V5-DEST expression vector to offset the requirement for Tat in the efficient production of viral RNA (Dull et al., 1998).

Biosafety Level 2 - Caution

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 miRNA targeting human genes involved in controlling cell division (e.g. tumor suppressor genes). 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).

Important

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™ HiPerform™ Lentiviral Pol II miR RNAi Expression System with EmGFP.

Cloning miR RNAi

Introduction

You will need to clone your miR RNAi sequence of interest contained within the engineered pre-miRNA into the pcDNA™6.2-GW/EmGFP-miR expression vector using the BLOCK-iT™ Pol II miR RNAi Expression Vector Kits to generate an expression clone prior to expressing your miR RNAi sequence of interest from pLenti6.4/R4R2/V5-DEST. After generating the expression clone, you will transfer the pre-miRNA expression cassette from the expression clone into the destination vector, pLenti6.4/R4R2/V5-DEST using a Rapid BP/LR recombination reaction.

General guidelines for cloning are provided below.

Using pcDNA6.2-GW/EmGFP-miR

To generate an expression clone in pcDNA™6.2-GW/EmGFP-miR, you will:

• Design and synthesize two complementary oligonucleotides containing your miRNA target sequence according to specified guidelines

• Anneal the oligonucleotides to create a double-stranded oligonucleotide

• Clone the double-stranded oligonucleotide into pcDNA™6.2-GW/EmGFPmiR using an optimized 5-minute ligation procedure

• Transform competent E. coli and select for expression clones

For detailed instructions and guidelines to generate your expression clone, refer to the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit manual. This manual is supplied with the kits and is also available for downloading from our web site (www.lifetechnologies.com) or by contacting Technical Support.

Using the pENTR™ 5’ Promoter Clone

Introduction

This section provides information on using the pENTR™ 5’ promoter clones.

CMV Promoter

pENTR™ 5’/CMVp contains the human CMV immediate early promoter to allow high-level, constitutive expression of the gene of interest in mammalian cells (Andersson et al., 1989; Boshart et al., 1985; Nelson et al., 1987). Although highly active in most mammalian cell lines, activity of the viral promoter can be downregulated in some cell lines due to methylation (Curradi et al., 2002), histone deacetylation (Rietveld et al., 2002), or both.

EF-1 Promoter

• Choice of human CMV immediate early promoter or EF-1 promoter

• attL4 and attR1 sites to allow two-fragment or three-fragment recombination with appropriate entry clone(s) and a MultiSite Gateway® destination vector to generate an expression construct

• Primer binding sites within the attL4 and attR1 sites for sequencing using the GW1 and GW3 primers

• rrnB transcription termination sequences to prevent basal expression of the PCR product of interest in E. coli

• Kanamycin resistance gene for selection in E. coli

• pUC origin for high-copy replication of the plasmid in E. coli

Cloning your own Promoter

To clone your own promoter, you will need the pENTR™ 5’/TOPO™ TA Cloning Kit . For details on how to clone your own promoter, refer to the pENTR™ 5’/TOPO™ TA Cloning Kit manual, which is available for downloading at www.lifetechnologies.com, or by contacting Technical Support

1. Perform the BP recombination reaction using the attB-expression clone with miR RNAi of interest and attP-containing pDONR™221 vector to produce a pENTR™221/EmGFP-miR entry clone.


2. Mix an aliquot of the BP reaction (containing the entry clone) with the pENTR™5’ promoter vector (attL4-attR1) and pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector (attR4-attR2) perform the LR recombination reaction to produce a lentiviral expression clone.


3. Transform the reaction mixture into a suitable E. coli host.


4. Select for lentiviral expression clones (see next page for a diagram of the recombination region of expression clones in pLenti6.4/R4R2/V5-DEST).

• Linearized attB-containing expression clones (see the next page for guidelines to linearize attB expression clones)
• attP-containing donor (pDONR™221) vector (see below) To perform an LR recombination reaction, you need the following substrates:
• Supercoiled attL1-attL2 entry vector (pENTR™221/EmGFP-miR)
• Supercoiled attL4-attR1 5’ entry vector (pENTR™5’/CMVp, pENTR™5’/EF-1αp, or other)
• Supercoiled attR4-attR2 destination vector (pLenti6.4/R4R2/V5-DEST)

1. Linearize 1-2 μg of the expression clone with a restriction enzyme (Eag I or BsrD I) that does not digest within the region of interest and is located outside the attB region.


2. Ethanol precipitate the DNA after digestion by adding 0.1 volume of 3 M sodium acetate followed by 2.5 volumes of 100% ethanol.


3. Pellet the DNA by centrifugation. Wash the pellet twice with 70% ethanol.


4. Dissolve the DNA in TE Buffer, pH 8.0 to a final concentration of 50-150 ng/μl.

1. Perform a BP recombination reaction using the linearized expression clone containing your pre-miRNA sequence and pDONR™221 to generate the entry clone, pENTR™221/EmGFP-miR.


2. Use a small aliquot of the BP reaction mix to perform the LR recombination reaction using a pENTR™5’ promoter clone (CMVp, EF-1αp, or other) and the pLenti6.4/R4R2/V5-DEST destination vector to generate the lentiviral expression clone, pLenti6.4/promoter/MSGW/EmGFP-miR.


3. Perform Proteinase K treatment.

• Linearized expression clone (50-150 ng/μl in TE Buffer, pH 8.0, see page 25)
• pDONR™221 vector (resuspend to 150 ng/μl in sterile water)
• pENTR™5’/CMVp or pENTR™5’/EF-1αp or your own pENTR™5’ entry clone
• pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector (150 ng/μl in TE Buffer, pH 8.0)
• pcDNA™6.2-GW/EmGFP-miR-neg control (if desired, supplied with BLOCKiT™ Pol II miR RNAi Expression Vector Kit with EmGFP, Box 1)
• BP Clonase™ II enzyme mix (supplied with the kit, Box 9; store at -20°C until immediately before use)
• LR Clonase™ II Plus enzyme mix (supplied with the kit, Box 10; store at -20°C until immediately before use)
• 2 μg/μl Proteinase K solution (supplied with Clonase™ enzymes; 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

1. Add the following components to sterile 0.5 ml microcentrifuge tubes at room temperature and mix.



Component	Sample	Positive Control
Linearized attB expression clone, (20-50 fmol)	1-7 μl	--
pcDNA™6.2-GW/EmGFPmiR-neg control (diluted to 50 ng/μl)	--	2 μl
pDONR™221 vector (150 ng/μl)	1 μl	1 μl
TE Buffer, pH 8.0	to 8 μl	5 μl




2. Remove the BP Clonase™ II enzyme mix from -20°C and thaw on ice (~ 2 minutes).


3. Vortex the BP Clonase™ II enzyme mix briefly twice (2 seconds each time).


4. To the samples above, add 2 μl of BP Clonase™ II enzyme mix. Mix well by pipetting up and down.
   
   Reminder:   Return BP Clonase™ II enzyme mix to -20°C immediately after use.


5. Incubate the reaction at 25°C for 1 hour. Important: Unlike the standard BP reaction, do not add Proteinase K to the samples. Instead, proceed immediately to the next step.


6. Transfer 3 μl from each BP reaction from Step 5 to clean, sterile 0.5 ml microcentrifuge tubes. This reaction mix contains the resulting entry clone, pENTR™221/EmGFP-miR.
   
   Note:   Save the remaining BP reaction mix at -20ºC for up to 1 week. You can treat the samples with Proteinase K and transform the reaction mix into One Shot® TOP10 Chemically Competent E. coli as described on page 61 to check the efficiency of the BP reaction. This also allows you to isolate entry clones for future use. For transformation of the BP reaction only, you can use any E. coli including TOP10.


7. Add the following components to the tubes containing 3 μl BP reaction from Step 6 at room temperature and mix.
   
   

   Component	Sample	Positive Control
   pENTR™5’ promoter vector (e.g. pENTR™5’/CMVp) (150 ng/μl)	1 μl	1 μl
   pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector (150 ng/μl)	1 μl	1 μl
   TE Buffer, pH 8.0	3 μl	3 μl

   
    


8. Remove the LR Clonase™ II Plus enzyme mix from -20°C and thaw on ice (~ 2 minutes).


9. Vortex the LR Clonase™ II Plus enzyme mix briefly twice (2 seconds each time).


10. To the samples above, add 2 μl of LR Clonase™ II Plus enzyme mix. Mix well by pipetting up and down.
    Reminder: Return LR Clonase™ II Plus enzyme mix to -20°C immediately after use.


11. Incubate the reaction at room temperature (20-25°C) from 16 hours to overnight.


12. Add 1 μl of the Proteinase K solution to each reaction. Incubate for 10 minutes at 37ºC.


13. Proceed to Transforming One Shot® Stbl3™ Competent E. coli, next page. Note:  You may store the reaction at -20°C for up to 1 week before transformation, if desired.

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 (from Step 13, Step 7)
• 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. W  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 miR RNAi cassette from pcDNA™6.2-GW/EmGFP-miR into the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector preserves the orientation of the cassette. However, if you wish to sequence your pLenti6.4 expression construct, we recommend using the following primers.

Primer                                                            Sequence
CMV Forward (for CMVp)                            5′-CGCAAATGGGCGGTAGGCGTG-3′
T7 Promoter (for EF-1αp)                              5′-TAATACGACTCACTATAGGA-3′
V5(C-term) Reverse                                     5′-ACCGAGGAGAGGGTTAGGGAT-3′

Note: For your convenience, Invitrogen has a custom primer synthesis Support. For more information, see our web site (www.lifetechnologies.com) or contact Technical Support

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.

Introduction

Before you can create a stably transduced cell line expressing your miR RNAi, you will first need to produce a lentiviral stock (containing the packaged pLenti6.4 expression construct) by co-transfecting the optimized ViraPower™ Packaging Mix and your pLenti6.4/promoter/MSGW/EmGFP-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 pLenti6.4/promoter/MSGW/EmGFP-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™ HiPerform™ Lentiviral Pol II miR RNAi Kit 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™ HiPerform™ Lentiviral Pol II miR RNAi Kit and is also available from our Web site (www.lifetechnologies.com) or by calling Technical Support.

Note:  The 293FT Cell Line is available separately from Invitrogen

Recommendation

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:

• Make sure that cells are greater than 90% viable.
• 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 pLenti6.4/promoter/MSGW/EmGFP-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™ HiPerform™ Lentiviral Pol II miR RNAi Kit 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 (page x) 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 (page x) or CsCl gradient centrifugation. Resuspend the purified pLenti6.4/promoter/MSGW/EmGFP-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™ HiPerform™ Lentiviral Pol II miR RNAi Kit 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

Opti-MEM® I

To facilitate optimal formation of DNA-Lipofectamine™ 2000 complexes, we recommend using Opti-MEM® I  Reduced Serum Medium available from Invitrogen. For more information about Opti-MEM® I, see our web site
(www.lifetechnologies.com) or call Technical Support


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 pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector and either the pENTR™5’/CMVp or pENTR™5’/EF-1αp vector using the Rapid BP/LR recombination reaction as described in this manual.
• Use the resulting lentiviral expression construct, pLenti6.4/promoter/MSGW/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.

pLenti6.4/CMV/V5-MSGW/lacZ Positive Control

A pLenti6.4/CMV/V5-MSGW/lacZ positive control vector is included with the pLenti6.4/R4R2/V5-DEST MultiSite Gateway® vector for use as an expression control in the ViraPower™ Lentiviral Expression System. The β-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, DH5α™-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 DH5α).


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 pLenti6.4/CMV/V5-MSGW/lacZ positive control to generate a control lentiviral stock (expressing the LacZ protein).


6. Use the pLenti6.4/CMV/V5-MSGW/lacZ lentiviral control and the pLenti6.4/promoter/MSGW/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:

• pLenti6.4/promoter/MSGW/EmGFP-miR expression construct (0.1-3.0 μg/μl in sterile water or TE Buffer, pH 8.0)
• Positive controls (see previous page 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 (pre-warmed)
• 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 10⁸ cells at a multiplicity of infection (MOI) = 1.

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.

Recommendation

The recommended procedure to co-transfect 293FT cells differs from the traditional Lipofectamine™ 2000 transfection procedure in that you will:

• First prepare DNA:Lipofectamine™ 2000 complexes and add them to plates containing growth media, then
• Add the 293FT cells to the media containing DNA:Lipofectamine™ 2000 complexes, allow the cells to attach, and transfect overnight.

Using this procedure, we consistently obtain lentiviral stocks with titers that are 3 to 4-fold higher than lentiviral stocks generated using the traditional Lipofectamine™ 2000 transfection procedure (i.e. plating cells first followed by transfection with DNA:Lipofectamine™ 2000 complexes). You may use the traditional Lipofectamine™ 2000 transfection procedure, if desired, but keep in mind that the viral titer obtained may be lower (see Alternative Transfection Procedure).

Reverse Transfection Procedure

Follow the procedure below to cotransfect 293FT cells. We recommend including a negative control (no DNA, no Lipofectamine™ 2000) in your experiment to help evaluate your results.

1. For each transfection sample, prepare DNA-Lipofectamine™ 2000 complexes as follows:
   • In a sterile 5 ml tube, dilute 9 μg ViraPower™ Packaging Mix and 3 μg pLenti6.4/promoter/MSGW/EmGFP-miR expression plasmid DNA (12 μg total) in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently.
   • In a separate sterile 5 ml tube, mix Lipofectamine™ 2000 gently before use, then dilute 36 μl in 1.5 ml of Opti-MEM® I Medium without serum. Mix gently and incubate for 5 minutes at room temperature.
   • After the 5 minute incubation, combine the diluted DNA with the diluted Lipofectamine™ 2000. Mix gently.
   • Incubate for 20 minutes at room temperature to allow the DNA-Lipid complexes to form. The solution ma  appear cloudy, but this will not impede the transfection.





2. While DNA-lipid complexes are forming, trypsinize and count the 293FT cells. Resuspend the cells at a density of 1.2 x 106 cells/ml in growth medium containing serum (or Opti-MEM® I Medium containing serum).


3. Add the DNA-Lipofectamine™ 2000 complexes to a 10 cm tissue culture plate containing 5 ml of growth medium containing serum (or Opti-MEM® I Medium containing serum). Do not include antibiotics in the medium.


4. Add 5 ml of the 293FT cell suspension (6 x 10⁶ total cells) to the plate containing media and DNA-Lipofectamine™ 2000 complexes and mix gently by rocking the plate back and forth. Incubate the cells overnight at 37°C in a CO₂ incubator.


5. The next day, remove the media containing the DNA-Lipofectamine™ 2000 complexes and replace with complete culture 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: Expression of the VSV G glycoprotein causes 293FT cells to fuse, resulting in the appearance of multinucleated syncytia which is normal and does not affect lentivirus production.


6. Harvest virus-containing supernatants 48-72 hours post-transfection by removing medium to a 15 ml sterile, capped, conical tube. Note: Minimal differences in viral yield are observed whether supernatants are collected 48 or 72 hours post-transfection.
   
   Caution:   Remember that you are working with infectious virus at this stage.


7. Centrifuge at 3000 rpm for 5 minutes at +4°C to pellet cell debris. Perform filtration step, if desired (see Note below).


8. Pipet viral supernatants into cryovials in 1 ml aliquots. Store viral stocks at -80°C.

Alternative (Forward) Transfection Procedure

An alternative (Forward) transfection procedure is provided below to cotransfect 293FT cells. Note that use of this procedure generally results in production of lentiviral stocks with a slightly lower titer that those produced when using the Recommended Transfection Procedure, above.

1. The day before transfection, plate the 293FT cells in a 10 cm tissue culture plate such that they will be 90-95% confluent on the day of transfection (i.e. 6 x 10⁶ cells in 10 ml of growth medium containing serum).


2. On the day of transfection, remove the culture medium from the 293FT cells and replace with 5 ml of growth medium containing serum (or Opti-MEM® I Medium containing serum). Do not include antibiotics in the medium.


3. Prepare DNA-Lipofectamine™ 2000 complexes as instructed in the Recommended Transfection Procedure, Step 1, above.


4. Add the DNA-Lipofectamine™ 2000 complexes dropwise to each plate of cells. Mix gently by rocking the plate back and forth. Incubate the cells overnight at 37°C in a CO₂ incubator.


5. Follow Steps 5-8 as instructed in the Recommended Transfection Procedure, above.

Note:  If you plan to use your lentiviral construct for in vivo applications, we recommend filtering your viral supernatant through a sterile, 0.45 μm low protein binding filter after the low-speed centrifugation step (see Step 7, above) to remove any remaining cellular debris. We recommend using Millex-HV 0.45 μm PVDF filters (Millipore, Catalog no. SLHVR25LS) for filtration. If you wish to concentrate your viral stock to obtain a higher titer, perform the filtration step first before concentrating your viral stock.

Long-Term Storage

Place lentiviral stocks at -80°C for long-term storage. Repeated freezing and thawing is not recommended as it may result in loss of viral titer. When stored properly, viral stocks of an appropriate titer should be suitable for use for up to one year. After long-term storage, we recommend re-titering your viral stocks before transducing your mammalian cell line of interest.

Scaling Up Virus Production

It is possible to scale up the cotransfection experiment to produce a larger volume of lentivirus, if desired. For example, we have scaled up the cotransfection experiment from a 10 cm plate to a T-175 cm2 flask and harvested up to 30 ml of viral supernatant. If you wish to scale up your cotransfection, remember that you will need to increase the number of cells plated and the amounts of DNA, Lipofectamine™ 2000, and medium used in proportion to the difference in surface area of the culture vessel.

• 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.

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

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.

• 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 ha  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 (see page for recommended storage conditions).

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

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.

• Your Lenti4/promoter/MSGW/EmGFP-miR 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)

• 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.

1. The day before transduction (Day 1), trypsinize and count the cells, plating them in a 6-well plate such that they will be 30-50% confluent at the time of transduction. Incubate cells at 37°C overnight. Example: When using HT1080 cells, we usually plate 2 x 10⁵ cells/well in a 6-well plate.


2. On the day of transduction (Day 2), thaw your lentiviral stock and prepare 10- fold serial dilutions ranging fro  10-2 to 10-6. For each dilution, dilute the lentiviral construct into complete culture medium to a final volume of 1 ml. DO NOT vortex.

Note:   You may prepare a wider range of serial dilutions (10-2 to 10-8), if desired.

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


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


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


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


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


8. Using a flow cytometry system, determine the percentage of GFP-positive cells for each dilution, see next page. Determine the titer using the formula described on the next page.


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


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


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


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


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


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

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.

Introduction

Once you have generated a lentiviral stock with a suitable titer, you are ready to transduce the lentiviral construct into your mammalian cell line to express the miR RNAi of interest and perform RNAi analysis. Guidelines are provided below. Reminder:   Remember that your lentiviral construct contains a deletion in the 3′ LTR that leads to self-inactivation of the lentivirus after transduction into mammalian cells. Once integrated into the genome, the lentivirus can no longer produce packageable virus.


Experimental Outline

To perform transduction, you will:

1. Determine the Multiplicity of Infection (MOI) and antibiotic sensitivity for your cell line.


2. Grow the mammalian cell line of choice.


3. Transduce the mammalian cell line of choice with your lentiviral construct in the presence of Polybrene®.


4. Harvest cells after 48-96 hours to perform transient knockdown experiments or select for stably transduced cells using Blasticidin.


5. Expand at least 5 Blasticidin-resistant colonies and analyze each clone to assay for knockdown of the target gene.

Factors Affecting Gene Knockdown Levels

A number of factors can influence the degree to which expression of your gene of interest is reduced (i.e. gene knockdown) in an RNAi experiment including:

• Transduction efficiency
• MOI used to transduce cells
• Transcription rate of the target gene of interest
• Stability of the target protein
• Growth characteristics of your mammalian cell line
• Activity of your miR RNAi in transient transfections

Take these factors into account when designing your transduction and RNAi experiments.


Transient vs. Stable Expression

After transducing your lentiviral construct into the mammalian cell line of choice, you may assay for target gene knockdown in the following ways:

• Pool a heterogeneous population of cells and test for gene knockdown directly after transduction (i.e. “transient” RNAi analysis). Note that you must wait for a minimum of 48-72 hours after transduction before harvesting your cells to allow expressed miR RNAi sequences to accumulate in transduced cells.
• Select for stably transduced cells using Blasticidin. This requires a minimum of 10-12 days after transduction, but allows generation of clonal cell lines that stably express the miR RNAi sequence.

Determining Antibiotic Sensitivity for Your Cell Line

Before selecting for stably transduced cells, you must first determine the minimum concentration of Blasticidin required to kill your untransduced mammalian cell line (i.e. perform a kill curve experiment).  If you titered your lentiviral construct in the same mammalian cell line that you are using to generate a stable cell line, then you may use the same concentration of Blasticidin for selection that you used for titering.

Multiplicity of Infection (MOI)

To obtain optimal expression of your miR RNAi and therefore, the highest degree of target gene knockdown, you will need to transduce the lentiviral construct into your mammalian cell line of choice using a suitable MOI. MOI is defined as the number of virus particles per cell and generally correlates with the number of integration events and as a result, expression. Typically, miR RNAi expression levels increase as the MOI increases.

Determining the Optimal MOI

A number of factors can influence determination of an optimal MOI including the nature of your mammalian cell line (e.g. non-dividing vs. dividing cell type; see Note below), its transduction efficiency, and the nature of your target gene of interest. If you are transducing your lentiviral construct into the mammalian cell line of choice for the first time, we recommend using a range of MOIs (e.g. 0, 1, 5, 10, 50) to determine the MOI required to obtain the optimal degree of target gene knockdown.

Note:  In general, non-dividing cell types transduce lentiviral constructs less efficiently than actively dividing cell lines. If you are transducing your lentiviral construct into a non-dividing cell type, you may need to increase the MOI to achieve an optimal degree of target gene knockdown.


Preparing Mammalian Cells

Initiate your mammalian cell line of choice that will be used for transduction. Grow the cells in the appropriate medium. Cells should be >95% viable.

Positive Controls

If you have generated two positive control lentiviral constructs (pLenti6.4/promoter/MSGW/EmGFP-miR-lacZ control and pLenti6.4/CMV/V5-MSGW/lacZ control constructs) as described on page 36, you may use the controls
in cotransduction experiments to verify the lentiviral induced RNAi response in mammalian cells. For cotransductions, use a 3:1 MOI ratio of pLenti6.4/promoter/MSGW/EmGFPmiR-lacZ to pLenti6.4/CMV/V5-MSGW/lacZ expression clone. The β-galactosidase protein expressed from the pLenti6.4/CMV/V5-MSGW/lacZ control lentiviral construct is approximately 121 kDa in size. You may assay for β-galactosidase expression by western blot analysis using β-gal Antiserum (Catalog no. R901-25), activity assay FluoReporter® lacZ/Galactosidase Quantitation Kit (Catalog no. F-2905), or by staining the cells for activity using the β-Gal Staining Kit (Catalog no. K1465-01) for fast and easy detection of β-galactosidase expression.

Important

Remember that viral supernatants are generated by harvesting spent media containing virus from the 293FT producer cells. Spent media lacks nutrients and may contain some toxic waste products. If you are using a large volume of viral supernatant to transduce your mammalian cell line (e.g. 1 ml of viral supernatant per well in a 6-well plate), note that growth characteristics or morphology of the cells may be affected during transduction. These effects are generally alleviated after transduction when the media is replaced with fresh, complete media.

Concentrating Virus

It is possible to concentrate VSV-G pseudotyped lentiviruses using a variety of methods without significantly affecting their transducibility. If the titer of your lentiviral stock is relatively low (less than 5 x 105 TU/ml) and your experiment requires that you use a large volume of viral supernatant (e.g. a relatively high MOI), you may wish to concentrate your virus before proceeding to transduction. For details and guidelines to concentrate your virus, refer to published reference sources (Yee, 1999).

Materials Needed

You will need the following materials before starting:

• Your titered 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 (if selecting for stably transduced cells)

                                                                                                                                                                                                       TOP
Transduction Procedure

Follow the procedure below to transduce the mammalian cell line of choice with your lentiviral construct.

1. Plate cells in complete media as appropriate for your application. When determining the density at which to plate cells, remember to take into account the length of time cells will be cultured prior to performing RNAi analysis (e.g 48 hours vs. 120 hours).


2. On the day of transduction (Day 1), thaw your lentiviral stock and dilute (if necessary) the appropriate amount of virus (at a suitable MOI) 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.  Note:  If you are transducing cells with undiluted viral stock and are concerned about possible toxicity or growth effects caused by overnight incubation, it is possible to incubate cells for as little as 6 hours prior to changing medium.


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


6. The following day (Day 3), perform one of the following:
   • Harvest the cells and assay for inhibition of your target gene if you are performing transient expression experiments. If you wish to assay the cells at a later time, you may continue to culture the cells or replate them into larger-sized tissue culture formats as necessary.
   • Remove the medium and replace with fresh, complete medium containing the appropriate amount of Blasticidin to select for stably transduced cells. Proceed to Step 7.




7. Replace medium with fresh medium containing Blasticidin every 3-4 days until Blasticidin-resistant colonies can be identified (generally 10-12 days after selection).


8. Pick at least 5 Blasticidin-resistant colonies (see Note below) and expand each clone to assay for knockdown of the target gene.


Note: 


Performing RNAi Analysis



What You Should See


TOP

Introduction

Review the information in this section to troubleshoot your lentiviral expression experiments. To troubleshoot oligo design and cloning experiments, refer to the BLOCK-iT™ Pol II miR RNAi Expression Vector Kit manual.

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

Rapid BP/LR Reaction and Transformation

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.

Transduction and RNAi Analysis

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

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