Inducible shRNA Expression Vectors

The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit combines Invitrogen’s BLOCK-iT™ RNAi and T-REx™ technologies to facilitate tetracycline-regulated expression of a short hairpin RNA (shRNA) of interest from an H1/TO RNAi cassette for use in RNA interference (RNAi) analysis in mammalian cells. The kit provides a Gateway-adapted entry vector designed to allow efficient transient or stable, regulated expression of shRNA in dividing mammalian cells or easy transfer of the H1/TO RNAi cassette into other suitable Gateway destination vectors for other RNAi applications. For more information about the BLOCK-iT™ RNAi, T-REx™, and Gateway technologies, see below.

Advantages of the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit

Using the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit for vector-based expression of shRNA provides the following advantages:

• Provides a rapid and efficient way to clone double-stranded oligonucleotide (ds oligo) duplexes encoding a desired shRNA target sequence into an entry vector containing an RNA Polymerase III (Pol III)-driven expression cassette (i.e. H1/TO RNAi cassette) for use in RNAi analysis.
• The entry construct containing the H1/TO RNAi expression cassette may be directly transfected into mammalian cells expressing the Tet repressor to enable rapid, tetracycline-regulated screening of shRNA target sequences.
• The entry construct contains a Zeocin™ resistance marker to allow generation of stable cell lines that express the shRNA of interest upon tetracycline addition.
• The vector is Gateway-adapted to allow easy transfer of the H1/TO RNAi cassette into any appropriate expression system for other RNAi applications (e.g. lentiviral system for stable delivery of regulated shRNA in hard-to-transfect or non-dividing mammalian cells).

BLOCK-iT™ RNAi Technology

A variety of BLOCK-iT™ RNAi products are available from Invitrogen to facilitate RNAi analysis in mammalian and invertebrate systems. The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit and the BLOCK-iT™ U6 RNAi Entry Vector Kit (Catalog nos. K4920-00 and K4945-00, respectively) use a vector-based approach to allow efficient generation of RNAi cassettes for constitutive or regulated expression of shRNA molecules in mammalian cells. 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, see the RNAi Central application portal at www.lifetechnologies.com/rnai or contact Technical Service


The T-REx™ Technology

The T-REx™ Technology facilitates tetracycline-regulated expression of a gene of interest in mammalian cells through the use of regulatory elements from the E. coli Tn10-encoded tetracycline (Tet) resistance operon (Hillen and Berens, 1994; Hillen et al., 1983). Tetracycline regulation in the T-REx™ System is based on the binding of tetracycline to the Tet repressor and derepression of the promoter controlling expression of the gene of interest (Yao et al., 1998). The main components of the T-REx™ System include:

• An inducible expression construct to facilitate tetracycline-regulated expression of your gene of interest under the control of a hybrid promoter containing two tetracycline operator 2 (TetO2) sites.
• A regulatory expression construct that facilitates high-level, constitutive expression of the Tet repressor (TetR). In the T-REx™ System, expression of the TetR gene is controlled by the CMV promoter.
• Tetracycline for inducing expression.

When the inducible expression construct and the regulatory expression construct are present in the same mammalian cell, expression of your gene of interest is repressed in the absence of tetracycline and induced in its presence (Yao et al., 1998).


Gateway Technology

The 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 (e.g. H1/TO RNAi cassette) into multiple vector systems. To express your shRNA of interest using the pENTR™/H1/TO vector, simply:
 

1. Clone your ds oligo encoding the shRNA of interest into the pENTR™/H1/TO vector to generate an entry clone.


2. Choose one of the following options:

• Transfect your entry construct into Tet repressor (TetR)-expressing mammalian cells. Add tetracycline to transiently assay for target gene knockdown.
• Transfect the entry construct into TetR-expressing mammalian cells and use Zeocin™ selection to generate a stable cell line. Add tetracycline to assay for target gene knockdown.
• Perform an LR recombination reaction between the entry construct and a suitable Gateway destination vector to generate an expression clone for use in other RNAi applications.

For more 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 calling Technical Service.

Note:   The BLOCK-iT™ Inducible H1 Lentiviral RNAi System also contains the BLOCK-iT™ Inducible H1 Lentiviral RNAi System components and the BLOCK-iT™ Inducible H1 Lentiviral RNAi System manual.

Kit Components
 
The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit and the BLOCK-iT™ Inducible H1 Lentiviral RNAi System include the following components. For a detailed description of the contents of the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit, see below. For a detailed description of the contents of the BLOCK-iT™ Inducible H1 Lentiviral RNAi reagents, see the BLOCK-iT™ Inducible H1 Lentiviral RNAi System manual.

Shipping/Storage

The BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit and the BLOCK-iT™ Inducible H1 Lentiviral RNAi System are shipped as described below. Upon receipt, store each item as detailed below. For more detailed information about the BLOCK-iT™ Inducible H1 Lentiviral RNAi reagents supplied with the kit, refer to the BLOCK-iT™ Inducible H1 Lentiviral RNAi System manual.

Inducible H1 RNAi Entry Vector Reagents and Tetracycline
 
The following reagents are included with the Inducible H1 RNAi Entry Vector and Tetracycline box (Box 1). Store the tetracycline at -20 ° C, protected from light. Store the other reagents at -20 ° C.

Unit Definition of T4 DNA Ligase
 
One (Weiss) unit of T4 DNA Ligase catalyzes the exchange of 1 nmol 32P-labeled pyrophosphate into [λ/β-32P]ATP in 20 minutes at 37 ° C (Weiss et al., 1968). One unit is equal to approximately 300 cohesive-end ligation units.

Primer Sequences

The table below provides the sequence and the amount supplied of the primers included in the kit.

LacZ2.1 Control Oligo Sequences

The sequences of the lacZ2.1 control oligos are listed below. The lacZ2.1 control DNA oligos are annealed and are supplied in the kit as a 5 nM double-stranded oligo that is ready-to-use in the ligation reaction

One Shot TOP10 Reagents

The following reagents are included in the One Shot TOP10 Chemically Competent E. coli kit (Box 2). Transformation efficiency is > 1 x 10⁹ cfu/µg plasmid DNA. Store at -80° C.
 

Flow Chart

The figure below illustrates the major steps necessary to produce a pENTR™/H1/TO entry clone using the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit.

Introduction

To use the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit, you will first need to design two single-stranded DNA oligonucleotides; one encoding the target shRNA (“top strand” oligo) and the other its complement (“bottom strand” oligo). You will then anneal the top and bottom strand oligos to generate a double-stranded oligonucleotide (ds oligo) suitable for cloning into the pENTR™/H1/TO vector.
 
The design of the single-stranded oligonucleotides (ss oligos) is critical to the success of both the cloning procedure and ultimately, the RNAi analysis. General guidelines are provided in this section to help you choose the target sequence and to design the ss oligos. Note however, that simply following these guidelines does not guarantee that the shRNA will be effective in knocking down the target gene. For a given target gene, you may need to generate and screen multiple shRNA sequences to identify one that is active in gene knockdown studies. 
 
Recommendation
 
We recommend using Invitrogen’s RNAi Designer, an online tool to help you design and order shRNA sequences for any target gene of interest. The RNAi Designer incorporates the guidelines provided in this manual as well as other design rules into a proprietary algorithm to design shRNA sequences from a target sequence that are compatible for use in cloning into the pENTR™/H1/TO or pENTR™/U6 vectors. Alternatively, if you have identified a synthetic siRNA that is active in triggering knockdown of your target gene, the RNAi Designer will convert the siRNA into a suitable shRNA. To use the RNAi Designer, see www.lifetechnologies.com/rnai.
 
Factors to Consider

When designing the top and bottom strand single-stranded oligos, consider the following factors:
 
Top strand oligo

• Sequences required to facilitate directional cloning
• Transcription initiation site
• Sequences encoding the shRNA of interest (i.e. stem and loop sequences)

Bottom strand oligo

• Sequences required to facilitate directional cloning
• Sequences complementary to the top strand oligo

For more information about the sequence requirements for directional cloning, see below. For guidelines to choose the target, loop, and transcription initiation sequences, see below. For an example of ss oligo design, see below.

Sequences Required for Directional Cloning

To enable directional cloning of the ds oligo into pENTR™/H1/TO, you must add the following 4 nucleotides to the 5' end of the corresponding ss oligo:

• Top strand oligo: Add CACC to the 5' end of the oligo. The CACC is complementary to the overhang sequence, GTGG, in the pENTR™/H1/TO vector and constitutes the last 4 bases of the H1/TO promoter.
• Bottom strand oligo: Add AAAA to the 5' end of the oligo. The AAAA is complementary to the overhang sequence, TTTT, in the pENTR™/H1/TO vector and constitutes the first 4 bases of the Pol III terminator.

Structural Features of the shRNA

Reminder:    When designing the top strand oligo encoding the shRNA, remember that an shRNA generally contains the following structural features:

• A short nucleotide sequence derived from the target gene (i.e. target sequence), followed by
• A short loop and
• A short nucleotide sequence that is the reverse complement of the initial target sequence.

 
Note that upon transcription, the target sequence and its complement base pair to form the stem of the shRNA. For guidelines to choose the target and loop sequences, see below.

Choosing the Target Sequence

When performing RNAi analysis on a particular gene, your choice of target sequence can significantly affect the degree of gene knockdown observed. We recommend following the guidelines below when choosing your target sequence. Note that these are general recommendations only, and that exceptions may occur.
 
Length:  We recommend choosing a target sequence ranging from 19 to 29 nucleotides in length. Longer sequences may induce non-specific responses in mammalian cells.

Complexity:

• Make sure that the target sequence does not contain runs of more than three of the same nucleotide. In particular, avoid choosing a target sequence that contains runs of four thymidines (T’s) as this will result in early transcription termination.
• Choose a sequence with low GC content (~30-50% GC content is recommended).
• Avoid choosing a target sequence that is a known site for RNA-protein interaction.

Homology:    Make sure that the target sequence does not contain significant homology to other genes as this can increase off-target RNAi effects.

Orientation:  You may choose a target sequence encoding the sense sequence of the target mRNA or the antisense sequence. Thus, you can generate an shRNA in two possible orientations: sense sequence-loop-antisense sequence or antisense sequence-loop-sense sequence.
 
siRNA:    If you have identified a synthetic siRNA that is active in triggering knockdown of your target gene, try generating an shRNA using this same target sequence.
 
Loop Sequence

You may use a loop sequence of any length ranging from 4 to 11 nucleotides, although short loops (i.e. 4-7 nucleotides) are generally preferred. Avoid using a loop sequence containing thymidines (T’s) as they may cause early termination. This is particularly true if the target sequence (see the previous page) itself ends in one or more T nucleotides.

Note: We have included the following loop sequences in active shRNA molecules:

• 5' -CGAA-3' 
• 5' -AACG-3' 
• 5' -GAGA-3'

Transcription of the shRNA initiates at the first base following the end of the H1/TO promoter sequence. In the top strand oligo, the transcription initiation site corresponds to the first nucleotide following the four base pair CACC sequence added to permit directional cloning. We recommend initiating the shRNA sequence at an adenosine (A) or a guanosine (G). Note that transcription of the native H1 RNA initiates at an A. Initiating transcription at a C or T is generally not recommended as this may affect initiation efficiency and position. When choosing the transcription initiation site, you should also keep the following in mind:
 
Initiation at an A

• If A is the first base of the target sequence, you do not need to add the complementary T to the 3' end of the top strand oligo because the T will be supplied by the first base of the Pol III terminator. Similarly, if the first 2 or 3 bases of the target sequence are A’s, you may omit adding the complementary T’s to the 3' end of the top strand oligo. For an example, see Example 2 below.
• If A is not the first base of the target sequence, add an A to the 5' end of the top strand oligo. You may omit adding a complementary T to the 3' end of the top strand oligo as the T will be supplied by the first base of the Pol III terminator.

Initiation at a G

• If G is the first base of the target sequence, then add a complementary C to the 3' end of the top strand oligo.
• If G is not the first base of the target sequence, we recommend adding a G to the 5' end of the top strand oligo directly following the CACC overhang sequence. In this case, do not add the complementary C to the 3'  end of the top strand oligo. For an example, see Example 1 below. Note:   We have found that adding the complementary C in this situation can result in reduced activity of the shRNA.

If you plan to express the same shRNA from both the pENTR™/H1/TO vector and Invitrogen’s pENTR™/U6 vector (i.e. BLOCK-iT™ U6 RNAi Entry Vector Kit, Catalog no. K4945-00), we recommend initiating the shRNA sequence at a G as this is the preferred initiation site for the U6 promoter. Generating shRNA sequence that initiates at a G allows the shRNA to be compatible for cloning and expression from either pENTR™/H1/TO or pENTR™U6.
 
Do not add 5' phosphates to your ss oligos during synthesis. The phosphate groups necessary for ligation are present in the linearized pENTR™/H1/TO vector.


Example 1: ss Oligo Design

This example lists the sequences of top and bottom strand oligos encoding an shRNA targeting the lamin A/C gene. These particular ss oligos were annealed to generate a lamin ds oligo that was cloned into pENTR™/H1/TO. The resulting lamin H1/TO RNAi cassette was transferred into the pLenti4/BLOCK-iT™-DEST vector in an LR recombination reaction to generate the pLenti4-GW/H1/TO-laminshRNA construct supplied in the BLOCK-iT™ Inducible H1 Lentiviral RNAi System (Catalog no. K4925-00).


 


Example 2: ss Oligo Design

This example lists the sequences of top and bottom strand oligos encoding an shRNA targeting the lacZ gene. These particular ss oligos were annealed to generate the lacZ2.1 ds control oligo supplied in the kit. Note that in this shRNA sequence, the first 3 bases of the target sequence are A’s. Thus, the 3 corresponding T’s were omitted from the 3' end of the top strand oligo.




We generally order unpurified, desalted single-stranded oligos using Invitrogen’s custom primer synthesis service (see www.invitrogen.com for more information) The ss oligos obtained anneal efficiently and provide optimal cloning results. Note however, that depending on which supplier you use, the purity and quality of the ss oligos may vary. If you obtain variable annealing and cloning results using unpurified, desalted oligos, you may want to order oligos that are HPLC or PAGE-purified.
 
Cloning Site and Recombination Region of pENTR™/H1/TO

Use the diagram below to help you design suitable DNA oligonucleotides to clone into pENTR™/H1/TO after annealing. Note the following features in the diagram below:

• The pENTR™/H1/TO vector is supplied linearized between nucleotides 1935 and 1936. The linearized vector contains 4 nucleotide overhangs on each strand encoding the last 4 nucleotides of the H1/TO promoter and the first 4 nucleotides of the Pol III terminator. Note that the annealed double-stranded (ds) oligo must contain specific 4 nucleotide 5' overhangs on each strand as indicated.
• The shaded region corresponds to those DNA sequences that will be transferred from the entry clone into the Gateway destination vector (e.g. pLenti4/BLOCK-iT™-DEST) following recombination.

Note:   Following recombination with a Gateway destination vector, the resulting expression clone will contain an RNAi cassette consisting of the H1/TO promoter, shRNA sequence, and the Pol III terminator.
The sequence of pENTR™/H1/TO is available for downloading from our Web site (www.lifetechnologies.com) or by contacting Technical Service.  For a map of pENTR™/H1/TO, see the Appendix.

Introduction

Once you have synthesized the appropriate complementary single-stranded DNA oligos, you will anneal equal amounts of each single-stranded oligo to generate a double-stranded oligo (ds oligo). Guidelines and instructions are provided in this section.
 
Single-Stranded Oligos

Before beginning, make sure that you have synthesized the single-stranded oligos with the appropriate sequences required for cloning into the pENTR™/H1/TO vector and for annealing. See the figure below for an illustration.

• “Top strand” oligo: Make sure that this oligo contains the sequence, CACC, at the 5' end.
• “Bottom strand” oligo: Make sure that this oligo contains the sequence, AAAA, at the 5' end and is complementary to the top strand oligo. 

Amount of DNA Oligo to Anneal

You will anneal equal amounts of the top and bottom strand oligos to generate the ds oligos. We generally perform the annealing reaction at a final single-stranded oligo concentration of 50 µM. Annealing at concentrations lower than 50 µM can significantly reduce the efficiency. Note that the annealing step is not 100% efficient; at least half of the single-stranded oligos remain unannealed even at a concentration of 50 µM.
 
Resuspending the Oligos

If your single-stranded oligos are supplied lyophilized, resuspend them in water or TE Buffer to a final concentration of 200 µM before use.
 
Materials Needed

Have the following materials on hand before beginning:

• Your “top strand” single-stranded oligo (200 µM in water or TE Buffer)
• Your “bottom strand” single-stranded oligo (200 µM in water or TE Buffer)
• 10X Oligo Annealing Buffer (supplied with the kit, Box 1)
• DNase/RNase-Free Water (supplied with the kit, Box 1)
• 0.5 ml sterile microcentrifuge tubes
• 95° C water bath or heat block

Follow this procedure to anneal your single-stranded oligos to generate the ds oligo. Note that the final concentration of the oligo mixture is 50 µM.

1. In a 0.5 ml sterile microcentrifuge tube, set up the following annealing reaction at room temperature.



Reagent	Amount
“Top strand” DNA oligo (200 µM)	5 µl
“Bottom strand” DNA oligo (200 µM)	5 µl
10X Oligo Annealing Buffer	2 µl
DNase/RNase-Free Water	8 µl
Total volume	20 µl


 

2. Incubate the reaction at 95° C for 4 minutes.


3. Remove the tube containing the annealing reaction from the water bath or the heat block and set on your laboratory bench.


4. Allow the reaction mixture to cool to room temperature for 5-10 minutes. The single-stranded oligos will anneal during this time.


5. Place the sample in a microcentrifuge and centrifuge briefly (~5 seconds). Mix gently.


6. Remove 1 µl of the annealing mixture and dilute the ds oligo as directed in Diluting the ds Oligo.


7. Store the remainder of the 50 µM ds oligo mixture at -20° C.

Diluting the ds Oligo

To clone your ds oligo into pENTR™/H1/TO, you must dilute the 50 µM stock to a final concentration of 5 nM (i.e. 10,000-fold dilution). We generally perform two 100-fold serial dilutions, the first into DNase/RNase-free water and the second into the 1X Oligo Annealing Buffer supplied with the kit. Follow the procedure below to dilute the ds oligo.

1. Dilute the 50 µM ds oligo mixture (from Annealing Procedure, Step 5) 100-fold into DNase/RNase-free water (i.e. 1 µl of 50 µM ds oligo into 99 µl of DNase/RNase-free water) to obtain a final concentration of 500 nM. Vortex to mix thoroughly.


2. Dilute the 500 nM ds oligo mixture (from Step 1) 100-fold into 1X Oligo Annealing Buffer as follows to obtain a final concentration of 5 nM. Vortex to mix thoroughly. Store the remaining 500 nM ds oligo stock at -20° C.
 
500 nM ds oligo                                        1 µl
10X Oligo Annealing Buffer                   10 µl
DNase/RNase-free water                      89 µl
Total volume                                         100 µl
 

3. Aliquot the 5 nM ds oligo stock and store at -20° C.

 
The undiluted ds oligos are 10,000-fold more concentrated than the working concentration. When performing the dilutions, be careful not to cross-contaminate the different ds oligo stocks. Remember to wear gloves and change pipette tips after every manipulation.
 
Storing the ds Oligo

Once you have diluted your ds oligo, you should have three stocks of annealed ds oligo. Use each stock as follows: 

• 50 µM ds oligo (undiluted): Use this stock to prepare new diluted ds oligo stocks if existing stocks become denatured or cross-contaminated.
• 500 nM ds oligo (100-fold dilution): Use this stock for gel analysis (see Checking the Integrity of the ds Oligo).
• 5 nM ds oligo (10,000-fold dilution): Use this stock for cloning.

Store the three ds oligo stocks at -20° C .

When using the diluted ds oligo stock solutions (i.e. 100-fold or 10,000-fold diluted stocks), thaw the solutions on ice. Do not heat or allow the ds oligo solutions to reach greater than room temperature as this causes the ds oligos to melt. The concentration of the oligos in the diluted solutions is not high enough to permit re-annealing and instead favors the formation of intramolecular hairpin structures. These intramolecular hairpin structures will not clone into pENTR™/H1/TO. 
 
If your diluted ds oligo stock solution(s) is heated, discard the ds oligo solution and prepare new diluted stocks using the procedure above.
 
Note:    If the 50 µM ds oligo solution (undiluted stock) becomes heated, the oligos are sufficiently concentrated and may be re-annealed following the annealing procedure.
 
Checking the Integrity of the ds Oligo

You may verify the integrity of your annealed ds oligo using agarose gel electrophoresis, if desired. We suggest running an aliquot of the annealed ds oligo (5 µl of the 500 nM stock) and comparing it to an aliquot of each starting single-stranded oligo (dilute the 200 µM stock 4000-fold to 500 nM; use 5 µl for gel analysis). Be sure to include an appropriate molecular weight standard. We generally use the following gel and molecular weight standard:

• Agarose gel: 4% E-Gel (Invitrogen, Catalog no. G5000-04)
• Molecular weight standard: 10 bp DNA Ladder (Invitrogen, Catalog no. 10821-015)

What You Should See

When analyzing an aliquot of the annealed ds oligo reaction by agarose gel electrophoresis, we generally see the following:

•A detectable higher molecular weight band representing annealed ds oligo.
•A detectable lower molecular weight band representing unannealed single-stranded oligos. Note that this band is detected since a significant amount of the single-stranded oligo remains unannealed.

For an example of expected results obtained from agarose gel analysis, see below. If the band representing ds oligo is weak or if you do not see a band, see Troubleshooting, for tips to troubleshoot your annealing reaction.
 
The efficiency at which ss oligos anneal may vary depending on their sequence and length. When analyzing the annealed ds oligo reaction by agarose gel electrophoresis, evaluate the annealing efficiency and roughly estimate the percentage of annealed ds oligo produced by comparing the intensity of the higher molecular weight band (annealed ds oligo) to the lower molecular band (unannealed ss oligos). You will use this information when setting up your ligation reaction.
 
Example of Expected Results

In this experiment, two 47 bp oligos (top and bottom strand) were annealed (50 µM final concentration) using the reagents supplied in the kit and following the procedure to generate a ds control oligo. The annealing reaction was diluted 100-fold in water to a concentration of 500 nM. Aliquots of the diluted ds oligo (5 µl) and each corresponding single-stranded oligo (5 µl of a 500 nM stock) were analyzed on a 4% E-Gel.
 
Results:   The ds oligo annealing reaction shows a clearly detectable, higher molecular weight band that differs in size from each component single-stranded oligo. Remaining unannealed ss oligo is also detectable. In this reaction, we estimate that the efficiency of the annealing reaction was greater than 50%.
 
Note:   The agarose gel is non-denaturing; therefore, the single-stranded oligos do not resolve at the expected size due to formation of secondary structure.

• The T4 DNA Ligase and the 5X Ligation Buffer supplied with the kit have been optimized to permit ligation of the ds oligo into the pENTR™/H1/TO vector in 5 minutes at room temperature. T4 DNA Ligase preparations and reaction buffers available from other manufacturers may not be appropriate for use in this application.

• Note:   The T4 DNA Ligase and reaction buffer supplied in the BLOCK-iT™ Inducible H1 RNAi Kits is available separately from Invitrogen (Catalog no. 15224-017).

• •Traditional ligation reactions are performed at 16° C overnight. This is not recommended for this application. Follow the ligation procedure below.

• If your ss oligos have annealed efficiently (i.e. the intensity of the higher molecular weight band is greater than the intensity of the lower molecular weight band on an agarose gel), then use 1-2 µl of the 5 nM ds oligo stock in the ligation reaction.
• If your ss oligos anneal less efficiently (i.e. the intensity of the higher molecular is equivalent to or less than the intensity of the lower molecular weight band on an agarose gel), then increase the amount of the 5 nM ds oligo stock used in the ligation reaction from 1 µl up to 5 µl.

• Double-stranded oligo of interest (5 nM in 1X Oligo Annealing Buffer; thaw on ice before use)
• pENTR™/H1/TO, linearized (0.5 ng/µl, supplied with the kit, Box 1; thaw on ice before use)
• lacZ2.1 ds control oligo (if desired; 5 nM in 1X Oligo Annealing Buffer; thaw on ice before use)
• 5X Ligation Buffer (supplied with the kit, Box 1)
• DNase/RNase-Free Water (supplied with the kit, Box 1)
• T4 DNA Ligase (1 U/µl, supplied with the kit, Box 1)

• Note:  The presence of PEG and glycerol (supplied by the Ligation Buffer and the T4 DNA Ligase) will make the reaction mixture viscous. Be sure to mix the reaction thoroughly by pipetting up and down. Do not vortex.

• Note:  The incubation time may be extended up to 2 hours and may result in a higher yield of colonies.

• Note:  You may store the ligation reaction at -20° C overnight.

• Ligation reaction (from Step 3)
• One Shot TOP10 Chemically Competent E. coli (supplied with the kit, Box 2; one vial per transformation; thaw on ice immediately before use)
• S.O.C. Medium (supplied with the kit, Box 2; warm to room temperature)
• pUC19 positive control (supplied with the kit, Box 2; if desired)
• 42° C water bath
• LB plates containing 50 µg/ml kanamycin (two for each transformation; warm at 37° C for 30 minutes before use)  Alternative: You may use Low Salt LB plates containing 50 µg/ml Zeocin™ to select for transformants, if desired. Note that for Zeocin™ to be active, the salt concentration of the bacterial medium must be < 90 mM and the pH must be 7.5.
  

• LB plates containing 100 µg/ml ampicillin (if transforming the pUC19 control)
• 37° C shaking and non-shaking incubator

1. Add 2 µl of the ligation reaction (from Step 3), into a vial of One Shot TOP10 chemically competent E. coli and mix gently. Do not mix by pipetting up and down.


2. Incubate on ice for 5 to 30 minutes.

Note:   Longer incubations seem to have a minimal effect on transformation efficiency. The length of the incubation is at the user’s discretion.

3. Heat-shock the cells for 30 seconds at 42° C without shaking.


4. Immediately transfer the tubes to ice.


5. Add 250 µl of room temperature S.O.C. Medium.


6. Cap the tube tightly and shake the tube horizontally (200 rpm) at 37° C for 1 hour.


7. Spread 40-200 µl from each transformation 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. If you are transforming the pUC19 control, plate 20-100 µl of the transformation reaction on pre-warmed LB plates containing 100 µg/ml ampicillin.


8. An efficient ligation reaction may produce several hundred colonies. Pick 5-10 colonies for analysis (see Analyzing Transformants).

1. Pick 5-10 kanamycin-resistant colonies and culture them overnight in LB or SOB medium containing 50 µg/ml kanamycin or Low Salt LB medium containing 50 µg/ml Zeocin™.


2. Isolate plasmid DNA using your method of choice. To obtain pure plasmid DNA for automated or manual sequencing, we recommend using the PureLink™ HQ Mini Plasmid Purification Kit (Catalog no. K2100-01) or the S.N.A.P.™ MidiPrep Kit (Catalog no. K1910-01) available from Invitrogen.


3. Sequence each pENTR™/H1/TO entry construct (see below) to confirm the following:

• The presence and correct orientation of the ds oligo insert.
• The sequence of the ds oligo insert.

• Note:  Because of the small size of the ds oligo insert, we do not recommend using restriction enzyme analysis to screen transformants.

• Note:   Entry clones containing mutated ds oligo inserts generally elicit a poor RNAi response in mammalian cells. Identify entry clones with the correct ds oligo sequence and use these clones for your RNAi analysis.

• Use high-quality, purified plasmid DNA for sequencing. We recommend preparing DNA using Invitrogen’s PureLink HQ Mini Plasmid Purification Kit (Catalog no. K2100-01) or S.N.A.P.™ MidiPrep Kit (Catalog no. K1910-01).
• Add DMSO to the sequencing reaction to a final concentration of 5%.
• Increase the amount of template used in the reaction (up to twice the normal concentration).
• Standard sequencing kits typically use dITP in place of dGTP to reduce G:C compression. Other kits containing dGTP are available for sequencing G-rich and GT-rich templates. If you are using a standard commercial sequencing kit containing dITP, obtain a sequencing kit containing dGTP (e.g. dGTP BigDye Terminator v3.0 Cycle Sequencing Ready Reaction Kit, Applied Biosystems, Catalog no. 4390229) and use a 7:1 molar ratio of dITP:dGTP in your sequencing reaction.

1. Streak the original colony out for a single colony on an LB plate containing 50 µg/ml kanamycin or a Low Salt LB plate containing 50 µg/ml Zeocin™.


2. Isolate a single colony and inoculate into 1-2 ml of LB containing 50 µg/ml kanamycin or Low Salt LB containing 50 µg/ml Zeocin™.


3. Grow until the culture reaches stationary phase.


4. Mix 0.85 ml of culture with 0.15 ml of sterile glycerol and transfer to a cryovial.


5. Store the glycerol stock at -80° C.

Introduction

Once you have generated your pENTR™/H1/TO entry construct, you are ready to express your shRNA of interest and to perform RNAi analysis of your target gene. This section provides general guidelines to help you design your transfection and RNAi experiment. We recommend that you read through this section before beginning.
 
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:

• Transfection efficiency
• Transcription rate of the target gene of interest
• Stability of the target protein
• Growth characteristics of your mammalian cell line
• Efficacy of the shRNA of interest

Take these factors into account when designing your RNAi experiments.
 
shRNA Expression Options

A number of options exist to express your shRNA of interest in the mammalian cell line of choice for RNAi analysis. Choose the option that best fits your needs.

Expression of Tet Repressor (TetR)

Because tetracycline-regulated shRNA expression in the BLOCK-iT™ Inducible H1 RNAi System is based on a repression/derepression mechanism, the amount of Tet repressor that is expressed in the host cell line will determine the level of transcriptional repression of the Tet operator sequences in your pENTR™/H1/TO construct. Tet repressor levels need to be sufficiently high to suitably repress basal level transcription of the shRNA, thus suppressing target gene knockdown in uninduced cells. In addition, the most effective repression of basal shRNA expression is achieved when Tet repressor is present in mammalian cells prior to introduction of the pENTR™/H1/TO construct.

For these reasons, we recommend first generating a stable cell line expressing the Tet repressor, then using this cell line as the host for your pENTR™/H1/TO entry construct (Option 2), or other suitable inducible expression construct (Option 3 above.)  This option is particularly recommended if you want to:

• Perform regulated RNAi knockdown experiments with several shRNA expression constructs in the same mammalian cell line.
• Obtain the lowest levels of basal shRNA expression (i.e. lowest levels of target gene knockdown in the absence of tetracycline)

To obtain a TetR-expressing stable cell line from Invitrogen, see the Recommendation below. For guidelines to generate your own stable TetR-expressing cell line, see Generating a TetR-Expressing Host Cell Line.

Several T-REx™ cell lines that stably express the Tet repressor are available from Invitrogen .   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 pENTR™/H1/TO entry 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.

Methods of Transfection
For established cell lines (e.g. COS, A549), consult original references or the supplier of your cell line for the optimal method of transfection. Pay particular attention to media requirements, when to pass the cells, and at what dilution to split the cells. Further information is provided in Current Protocols in Molecular Biology (Ausubel et al., 1994).
 
Methods for transfection include calcium phosphate (Chen and Okayama, 1987; Wigler et al., 1977), lipid-mediated (Felgner et al., 1989; Felgner and Ringold, 1989), and electroporation (Chu et al., 1987; Shigekawa and Dower, 1988). Choose the method and reagent that provides the highest efficiency transfection in your mammalian cell line. For a recommendation, see below.
 
For high-efficiency transfection in a broad range of mammalian cell lines, we recommend using the cationic lipid-based Lipofectamine™ 2000 Reagent (Catalog no. 11668-027) available from Invitrogen (Ciccarone et al., 1999). Using Lipofectamine™ 2000 to transfect plasmid DNA into eukaryotic cells offers the following advantages:

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

For more information on Lipofectamine™ 2000 Reagent, refer to our Web site (www.lifetechnologies.com) or call Technical Service

Transient vs. Stable Expression of Your shRNA

When designing your RNAi experiment, you should consider how to assay for knockdown of the target gene. After you have transfected your pENTR™/H1/TO construct into TetR-expressing mammalian cells, you may:

• Pool a heterogeneous population of cells and test for target gene knockdown after induction with tetracycline (i.e. transient knockdown). We recommend waiting for a minimum of 24-48 hours after induction before assaying for target gene knockdown to allow time for the shRNA to be expressed and processed.
• Select for stably transfected cells using Zeocin™. Selection requires a minimum of 10-14 days after transfection, but allows generation of clonal cell lines that stably express the shRNA of interest. shRNA expression will be tetracycline-regulated. For more information about Zeocin™ selection, see Generating a Stable Cell Line.

Tetracycline

(MW = 444.4) is commonly used as a broad spectrum antibiotic and acts to inhibit translation by blocking polypeptide chain elongation in bacteria. In the BLOCK-iT™ Inducible H1 RNAi System, tetracycline functions as an inducing agent to regulate transcription of the shRNA of interest from the H1/TO RNAi cassette. Tetracycline is supplied with the BLOCK-iT™ Inducible H1 RNAi Kits as a 10 mg/ml stock solution that is ready-to-use, but is also available separately from Invitrogen in powdered form (Catalog no. Q100-19).

Using Tetracycline

To induce transcription of the shRNA of interest in mammalian cells, we generally add tetracycline to a final concentration of 1 µg/ml in complete growth medium. If desired, you may vary the concentration of tetracycline used for induction from 0.001 to 1 µg/ml to modulate expression of the shRNA of interest.
 
Note:   The concentrations of tetracycline used for induction in the BLOCK-iT™ Inducible H1 RNAi System are generally not high enough to be toxic to mammalian cells.
 
Follow the guidelines below when handling tetracycline.

• Tetracycline is light sensitive. Store the stock solution at -20° C, protected from light. Prepare medium containing tetracycline immediately before use.
• Tetracycline is toxic. Do not ingest solutions containing the drug. If handling the powdered form, do not inhale.
• Wear gloves, a laboratory coat, and safety glasses or goggles when handling tetracycline and tetracycline-containing solutions.

Tetracycline in Fetal Bovine Serum

When culturing cells in medium containing fetal bovine serum (FBS), note that many lots of FBS contain tetracycline as FBS is often isolated from cows that have been fed a diet containing tetracycline. If you culture your mammalian cells in medium containing FBS that is not reduced in tetracycline, you may observe some basal expression of your shRNA of interest in the absence of tetracycline. We generally culture our mammalian cells in medium containing FBS that may not be reduced in tetracycline, and have observed low basal expression of shRNA (as assayed by % target gene knockdown) in the absence of tetracycline. Depending on your application (e.g. if targeting a protein involved in cell viability), you may wish to culture your cells in tetracycline-tested FBS. You may obtain tetracycline-tested GIBCO FBS from Invitrogen.

Introduction

This section provides general guidelines to transfect your pENTR™/H1/TO construct into a TetR-expressing mammalian cell line of interest to perform transient, regulated RNAi analysis. Performing transient RNAi analysis is useful to:

• Quickly test multiple shRNA sequences to a particular target gene
• Quickly screen for an RNAi response in your mammalian cell line

If you want to generate a stable cell line expressing the shRNA of interest, see the next section.
 
Reminder:   For optimal results, we recommend that you transfect your pENTR™/H1/TO construct into a mammalian cell line that stably expresses high levels of the Tet repressor (i.e. use one of Invitrogen’s T-REx™ Cell Lines or a cell line that you have generated). If you have not generated a stable TetR-expressing cell line, you may co-transfect the pENTR™/H1/TO plasmid with a suitable TetR-expressing plasmid (i.e. pcDNA™6/TR or pLenti6/TR) into your mammalian cell line. If you wish to use this method, we recommend using 6-fold more TetR expression plasmid DNA than pENTR™/H1/TO plasmid DNA in the co-transfection. For example, use 600 ng of pcDNA™6/TR plasmid and 100 ng of pENTR™/H1/TO entry construct DNA when transfecting cells plated in a 24-well format. Note that you may need to optimize repression and inducibility by varying the ratio of TetR expression plasmid:pENTR™/H1/TO used for transfection.
 
Plasmid Preparation

Once you have obtained your entry 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 or 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™ HQ Mini Plasmid Purification Kit (Catalog no. K2100-01), S.N.A.P.™ MidiPrep Kit (Catalog no. K1910-01) or CsCl gradient centrifugation.
 
Positive Control

If you have performed the positive control reaction and have cloned the lacZ2.1 ds oligo supplied with the kit into pENTR™/H1/TO, we recommend using the resulting pENTR™-GW/H1/TO-lacZ2.1shRNA entry construct as a positive control to assess the RNAi response in your cell line. Simply co-transfect the pENTR™-GW/H1/TO-lacZ2.1shRNA entry construct and the pcDNA™1.2/V5-GW/lacZ reporter plasmid supplied with the kit into your TetR-expressing mammalian cells and assay for knockdown of b-galactosidase expression 48 hours post-transfection using Western blot analysis or activity assay. For more information about the pcDNA™1.2/V5-GW/lacZ reporter plasmid, recommendations for transfection, and methods to assay for b-galactosidase activity, see below.

pcDNA™1.2/V5-GW/lacZ Reporter Plasmid

The pcDNA™1.2/V5-GW/lacZ reporter plasmid is supplied with the kit for use as a positive control to assay for the RNAi response in your mammalian cell line. In this vector, ß-galactosidase is expressed as a C-terminally tagged fusion protein under the control of the human cytomegalovirus (CMV) promoter.
 
The pcDNA™1.2/V5-GW/lacZ vector is supplied as 10 µg of plasmid DNA, lyophilized in TE Buffer, pH 8.0. To use the vector, resuspend in 10 µl of sterile water to obtain a 1 µg/µl stock. Dilute the stock as necessary for use in transfection (see  below).  If you wish to propagate the plasmid, transform a recA, endA E. coli strain such as TOP10. Use 10 ng of plasmid for transformation and select on LB agar plates containing 100 µg/ml ampicillin.

Transfecting the LacZ-Containing Reagents

To perform RNAi analysis using the lacZ control reagents, you will co-transfect the pcDNA™1.2/V5-GW/lacZ reporter plasmid and the pENTR™-GW/H1/TO-lacZ2.1shRNA entry construct that you have generated into your TetR-expressing mammalian cell line. For optimal results, we recommend using 6-fold more entry construct DNA than reporter plasmid DNA in the co-transfection. For example, use 600 ng of pENTR™-GW/H1/TO-lacZ2.1shRNA DNA and 100 ng of pcDNA™1.2/V5-GW/lacZ DNA when transfecting cells plated in a 24-well format.
 
Materials Needed

Have the following materials on hand before beginning:

• TetR-expressing mammalian cell line of interest (make sure that cells are healthy and > 90% viable before beginning)   Note:   If your cell line expresses TetR from pcDNA™6/TR or pLenti6/TR, maintain the cells in medium containing the appropriate concentration of Blasticidin.
• pENTR™/H1/TO entry construct
• pcDNA™1.2/V5-GW/lacZ plasmid (if performing the positive control transfection; supplied with the kit, Box 1, resuspend in water to 1 µg/µl)
• pENTR™-GW/H1/TO-lacZ2.1shRNA plasmid (if you have performed the positive control ligation reaction and are performing the positive control transfection)
• Transfection reagent of choice (e.g. Lipofectamine™ 2000)
• Tetracycline (supplied with the kit, Box 1; 10 mg/ml stock solution)
• Appropriate tissue culture dishes and supplies

Guidelines are provided below to transfect your pENTR™/H1/TO entry construct into the TetR-expressing mammalian cell line of choice and to induce expression of the shRNA of interest with tetracycline.
 

1. One day before transfection, plate cells at a density recommended by the manufacturer of the transfection reagent you are using.


2. On the day of transfection (Day 1), transfect your pENTR™/H1/TO construct into cells following the recommendations of the manufacturer of your transfection reagent. If you are co-transfecting the pENTR™/H1/TO construct and a TetR expression plasmid or the pcDNA™1.2/V5-GW/lacZ and pENTR™-GW/H1/TO-lacZ2.1shRNA plasmids, use the appropriate amounts of each plasmid as recommended.


3. At an appropriate time (generally 3 to 24 hours) after transfection, remove medium and replace with fresh growth medium containing 1 µg/ml tetracycline to induce shRNA expression. Note the following:


• If you have transfected your cells using Lipofectamine™ 2000, you may add tetracycline to induce expression of your shRNA as early as 3 hours following transfection.
• If you have included the lacZ positive control plasmids in your experiment, add tetracycline to cells 3 hours after transfection. This induces expression of the lacZ2.1 shRNA and prevents accumulation of ß-galactosidase, enabling detectable measurement of lacZ knockdown that might otherwise be masked by the long half-life of ß-galactosidase.
• If you have transfected your cells using another transfection reagent, you may need to replace the medium and allow cells to recover for 24 hours before induction.



4. Incubate cells in medium containing tetracycline for 24 to 96 hours, as appropriate before assaying for target gene knockdown.

 
Assaying for ß-galactosidase Expression

If you perform RNAi analysis using the control entry clone containing the lacZ2.1 ds oligo (i.e. pENTR™-GW/H1/TO-lacZ2.1shRNA), you may assay for ß-galacto-sidase expression and knockdown by Western blot analysis or activity assay using cell-free lysates (Miller, 1972). Invitrogen offers the ß-gal Antiserum (Catalog no. R901-25), the ß-Gal Assay Kit (Catalog no. K1455-01), and the FluoReporter lacZ/Galactosidase Quantitation Kit (Catalog no. F-2905) for detection of ß-galactosidase expression. For an example of results obtained from a ß-galacto-sidase knockdown experiment, see below.

Note:  The ß-galactosidase protein expressed from the pcDNA™1.2/V5-GW/lacZ control plasmid is fused to a V5 epitope and is approximately 119 kDa in size. If you are performing Western blot analysis, you may also use the Anti V5 Antibodies available from Invitrogen (e.g. Anti-V5-HRP Antibody; Catalog no. R961-25 or Anti-V5-AP Antibody, Catalog no. R962-25) for detection. For more information, refer to our Web site (www.invitrogen.com) or call Technical Service.

Example of Expected Results: Transient, Regulated Knockdown of a lacZ Reporter Gene

In this experiment, pENTR™/H1/TO entry constructs containing ds oligo encoding shRNA targeting the lacZ (i.e. pENTR™-GW/H1/TO-lacZ2.1shRNA) reporter gene or the endogenous lamin (i.e. pENTR™-GW/H1/TO-laminshRNA) gene were generated following the recommended protocols and using the reagents supplied in the BLOCK-iT™ Inducible H1 RNAi Entry Vector Kit. Note that the lacZ ds oligo used in this experiment is the same as the lacZ2.1 ds control oligo supplied with the kit.
 
T-REx™-293 cells (Invitrogen, Catalog no. R710-07) were grown to 90% confluence. Individual wells in a 24-well plate were transfected using Lipofectamine™ 2000 Reagent with 700 ng of plasmid DNA (100 ng of the pcDNA™1.2/V5-GW/lacZ reporter plasmid and 600 ng of non-specific plasmid DNA). In some wells, the reporter plasmid was co-transfected with 600 ng of the pENTR™-GW/H1/TO-lacZ2.1shRNA or pENTR™-GW/H1/TO-laminshRNA constructs. Three hours after transfection, the medium was replaced with medium containing 1 µg/ml tetracycline. Cell lysates were prepared 48 hours after induction and assayed for ß-galactosidase activity.
 
Results:  Potent and specific inhibition of b-galactosidase activity is evident from the lacZ-derived shRNA but not from the lamin-derived shRNA after cells have been treated with tetracycline.
 
Note:   In this experiment, some basal expression of the lacZ-derived shRNA occurs as evidenced by the ~ 15% inhibition of ß-galactosidase activity in cells cultured in the absence of tetracycline.

• Vast increase in size (similar to the effects of cytomegalovirus infecting permissive cells)
• Abnormal cell shape
• Presence of large empty vesicles in the cytoplasm (breakdown of the endoplasmic reticulum and Golgi apparatus or scaffolding proteins)
• Breakdown of plasma and nuclear membrane (appearance of many holes in these membranes)
• Eventually, these “cells” will completely break down and only “strings” of protein will remain.

• TetR-expressing mammalian cell line of interest (make sure that cells are healthy and > 90% viable before beginning)   Note:   If your cell line expresses TetR from pcDNA™6/TR or pLenti6/TR, maintain the cells in medium containing the appropriate concentration of Blasticidin.
• pENTR™/H1/TO entry construct
• Transfection reagent of choice (e.g. Lipofectamine™ 2000)
• Zeocin™ (100 mg/ml in sterile water)
• Blasticidin (to maintain the pcDNA™6/TR or pLenti6/TR construct) in the TetR-expressing cell line
• Tetracycline (supplied with the kit, Box 1; 10 mg/ml stock solution)
• Appropriate tissue culture dishes and supplies

1. One day before transfection, plate cells at a density recommended by the manufacturer of the transfection reagent you are using.


2. On the day of transfection (Day 1), transfect your pENTR™/H1/TO construct into cells following the recommendations of the manufacturer of your transfection reagent.


3. Four to six hours after transfection, remove the medium and replace with fresh growth medium. Incubate the cells overnight at 37°  C.


4. The following day (Day 2), trypsinize and replate cells into a larger-sized tissue culture format in fresh complete medium containing the appropriate concentrations of Blasticidin and Zeocin™. Note: Blasticidin is required to maintain the pcDNA™6/TR or pLenti6/TR construct in the TetR-expressing cells. Example: If transfecting cells in a 6-well format, trypsinize and replate cells into a 10 cm tissue culture plate in medium containing Blasticidin and Zeocin™.


5. Replace medium with fresh medium containing Blasticidin and Zeocin™ every 3-4 days until Blasticidin- and Zeocin™-resistant colonies can be identified (generally 10-14 days after selection).


6. Pick at least 10 Blasticidin- and Zeocin™-resistant colonies and expand each clone.


7. 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. Compare to uninduced cells.

• Perform delivery of the regulated shRNA of interest to “hard-to-transfect” or non-dividing mammalian cells. Use the pLenti4/BLOCK-iT™-DEST vector (see Note below).
• Generate stable cell lines expressing the regulated shRNA using a selection marker other than Zeocin™. Use the pBLOCK-iT™3-DEST vector containing the neomycin selection marker (Catalog no. V486-20).

• Supercoiled attL-containing pENTR™/H1/TO entry clone
• Supercoiled attR-containing destination vector

• Purified plasmid DNA of your pENTR™/H1/TO entry clone (50-150 ng/µl in TE Buffer, pH 8.0)
• Destination vector of choice (150 ng/µl in TE Buffer, pH 8.0)
• LR Clonase™ II enzyme mix (Invitrogen, Catalog no. 11791-020)
• TE Buffer, pH 8.0 (10 mM Tris-HCl, pH 8.0, 1 mM EDTA)
• 2 µg/µl Proteinase K solution (supplied with the LR Clonase™ II enzyme mix)
• Appropriate chemically competent E. coli host and growth media for expression
• S.O.C. Medium
• Appropriate selective plates

Introduction

Use the information in this section to troubleshoot the annealing, cloning, transformation, and transfection procedures.
 
Annealing Reaction

The table below lists some potential problems and possible solutions that may help you troubleshoot the annealing reaction.

Ligation and Transformation Reactions

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

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.

pENTR™/H1/TO Map

The complete nucleotide sequence of this vector is available for downloading at www.invitrogen.com or by contacting Technical Service.

Features of pENTR™/H1/TO
 
pENTR™/H1/TO (3869 bp) contains the following elements. All features have been functionally tested and the vector fully sequenced.

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.

1. For 1 liter, dissolve 10 g tryptone, 5 g yeast extract, and 5 g NaCl in 950 ml deionized water.


2. Adjust the pH of the solution to 7.5 with NaOH and bring the volume up to 1 liter. If preparing plates, add 15 g/L agar.


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. If preparing plates, pour into 10 cm plates.


4. Store at +4°C. Plates containing Zeocin™ may be stored at +4°C for up to 2 weeks.

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.

• Transfect the pcDNA™6/TR plasmid (i.e. TetR expression plasmid from the T-REx™ System) into your mammalian cells of interest. Use Blasticidin to select for a stable cell line.
• Transfect the pLenti6/TR plasmid (i.e. TetR expression plasmid from the ViraPower™ T-REx™ and BLOCK-iT™ Inducible H1 Lentiviral RNAi System) into your mammalian cells of interest. Alternatively, produce a Lenti6/TR lentiviral stock, and use this stock to transduce the mammalian cells of interest. Use Blasticidin to select for a stable cell line.

• Maintain the cell line in medium containing Blasticidin
• Remember to freeze and store vials of early passage cells 

1. Ambros, V. (2001). MicroRNAs: Tiny Regulators with Great Potential. Cell 107, 823-826.


2. Anandalakshmi, R., Pruss, G. J., Ge, X., Marathe, R., Mallory, A. C., Smith, T. H., and Vance, V. B. (1998). A Viral Suppressor of Gene Silencing in Plants. Proc. Natl. Acad. Sci. USA 95, 13079-13084.


3. Andersson, S., Davis, D. L., Dahlbäck, H., Jörnvall, H., and Russell, D. W. (1989). Cloning, Structure, and Expression of the Mitochondrial Cytochrome P-450 Sterol 26-Hydroxylase, a Bile Acid Biosynthetic Enzyme. J. Biol. Chem. 264, 8222-8229.


4. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1994). Current Protocols in Molecular Biology (New York: Greene Publishing Associates and Wiley-Interscience).


5. Baer, M., Nilsen, T. W., Costigan, C., and Altman, S. (1990). Structure and Transcription of a Human Gene for H1 RNA, the RNA Component of Human RNase P. Nuc. Acids Res. 18, 97-103.


6. Bernstein, E., Caudy, A. A., Hammond, S. M., and Hannon, G. J. (2001). Role for a Bidentate Ribonuclease in the Initiation Step of RNA Interference. Nature 409, 363-366.


7. Bogenhagen, D. F., and Brown, D. D. (1981). Nucleotide Sequences in Xenopus 5S DNA Required for Transcription Termination. Cell 24, 261-270.


8. Boshart, M., Weber, F., Jahn, G., Dorsch-Häsler, K., Fleckenstein, B., and Schaffner, W. (1985). A Very Strong Enhancer is Located Upstream of an Immediate Early Gene of Human Cytomegalovirus. Cell 41, 521-530.


9. Bosher, J. M., and Labouesse, M. (2000). RNA Interference: Genetic Wand and Genetic Watchdog. Nature Cell Biol. 2, E31-E36.


10. Brummelkamp, T. R., Bernards, R., and Agami, R. (2002). A System for Stable Expression of Short Interfering RNAs in Mammalian Cells. Science 296, 550-553.


11. Calmels, T., Parriche, M., Burand, H., and Tiraby, G. (1991). High Efficiency Transformation of Tolypocladium geodes Conidiospores to Phleomycin Resistance. Curr. Genet. 20, 309-314.


12. Carrington, J. C., and Ambros, V. (2003). Role of MicroRNAs in Plant and Animal Development. Science 301, 336-338.


13. Chen, C., and Okayama, H. (1987). High-Efficiency Transformation of Mammalian Cells by Plasmid DNA. Mol. Cell. Biol. 7, 2745-2752.


14. Chu, G., Hayakawa, H., and Berg, P. (1987). Electroporation for the Efficient Transfection of Mammalian Cells with DNA. Nucleic Acids Res. 15, 1311-1326.


15. Ciccarone, V., Chu, Y., Schifferli, K., Pichet, J.-P., Hawley-Nelson, P., Evans, K., Roy, L., and Bennett, S. (1999). LipofectamineTM 2000 Reagent for Rapid, Efficient Transfection of Eukaryotic Cells. Focus 21, 54-55.


16. Cogoni, C., and Macino, G. (1999). Gene Silencing in Neurospora crassa Requires a Protein Homologous to RNA-Dependent RNA Polymerase. Nature 399, 166-169.


17. Cogoni, C., and Macino, G. (1997). Isolation of Quelling-Defective (qde) Mutants Impaired in Posttranscriptional Transgene-Induced Gene Silencing in Neurospora crassa. Proc. Natl. Acad. Sci. USA 94, 10233-10238.


18. Cogoni, C., Romano, N., and Macino, G. (1994). Suppression of Gene Expression by Homologous Transgenes. Antonie Van Leeuwenhoek 65, 205-209.


19. Cole, C. N., and Stacy, T. P. (1985). Identification of Sequences in the Herpes Simplex Virus Thymidine Kinase Gene Required for Efficient Processing and Polyadenylation. Mol. Cell. Biol. 5, 2104-2113.


20. Curradi, M., Izzo, A., Badaracco, G., and Landsberger, N. (2002). Molecular Mechanisms of Gene Silencing Mediated by DNA Methylation. Mol. Cell. Biol. 22, 3157-3173.


21. Drocourt, D., Calmels, T. P. G., Reynes, J. P., Baron, M., and Tiraby, G. (1990). Cassettes of the Streptoalloteichus hindustanus ble Gene for Transformation of Lower and Higher Eukaryotes to Phleomycin Resistance. Nucleic Acids Res. 18, 4009.


22. Dykxhoorn, D. M., Novina, C. D., and Sharp, P. A. (2003). Killing the Messenger: Short RNAs that Silence Gene Expression. Nat. Rev. Mol. Cell Biol. 4, 457-467.


23. Felgner, P. L., Holm, M., and Chan, H. (1989). Cationic Liposome Mediated Transfection. Proc. West. Pharmacol. Soc. 32, 115-121.


24. Felgner, P. L. a., and Ringold, G. M. (1989). Cationic Liposome-Mediated Transfection. Nature 337, 387-388.


25. Grishok, A., Pasquinelli, A. E., Conte, D., Li, N., Parrish, S., Ha, I., Baillie, D. L., Fire, A., Ruvkun, G., and Mello, C. C. (2001). Genes and Mechanisms Related to RNA Interference Regulate Expression of the Small Temporal RNAs That Control C. elegans Developmental Timing. Cell 106, 23-34.


26. Hammond, S. M., Bernstein, E., Beach, D., and Hannon, G. J. (2000). An RNA-Directed Nuclease Mediates Genetic Interference in Caenorhabditis elegans. Nature 404, 293-296.


27. Hannon, G. J. (2002). RNA Interference. Nature 418, 244-251.


28. Hannon, G. J., Chubb, A., Maroney, P. A., Hannon, G., Altman, S., and Nilsen, T. W. (1991). Multiple cis-acting Elements are Required for RNA Polymerase III Transcription of the Gene Encoding H1 RNA, the RNA Component of Human RNase P. J. Biol. Chem. 266, 22796-22799.


29. Hillen, W., and Berens, C. (1994). Mechanisms Underlying Expression of Tn10 Encoded Tetracycline Resistance. Annu. Rev. Microbiol. 48, 345-369.


30. Hillen, W., Gatz, C., Altschmied, L., Schollmeier, K., and Meier, I. (1983). Control of Expression of the Tn10-encoded Tetracycline Resistance Genes: Equilibrium and Kinetic Investigations of the Regulatory Reactions. J. Mol. Biol. 169, 707-721.


31. Hutvagner, G., McLachlan, J., Pasquinelli, A. E., Balint, E., Tuschl, T., and Zamore, P. D. (2001). A Cellular Function for the RNA-Interference Enzyme Dicer in the Maturation of the let-7 Small Temporal RNA. Science 293, 811-813.


32. Jones, A. L., Thomas, C. L., and Maule, A. J. (1998). De novo Methylation and Co-Suppression Induced by a Cytoplasmically Replicating Plant RNA Virus. EMBO J. 17, 6385-6393.


33. Ketting, R. F., Fischer, S. E., Bernstein, E., Sijen, T., Hannon, G. J., and Plasterk, R. H. (2001). Dicer Functions in RNA Interference and in Synthesis of Small RNA Involved in Developmental Timing in C. elegans. Genes Dev. 15, 2654-2659.


34. Landy, A. (1989). Dynamic, Structural, and Regulatory Aspects of Lambda Site-specific Recombination. Ann. Rev. Biochem. 58, 913-949.


35. Lee, R. C., Feinbaum, R. L., and Ambros, V. (1993). The C. elegans Heterochronic Gene lin-4 Encodes Small RNAs with Antisense Complementarity to lin-14. Cell 75, 843-854.


36. Li, W. X., and Ding, S. W. (2001). Viral Suppressors of RNA Silencing. Curr. Opin. Biotechnol. 12, 150-154.


37. McManus, M. T., Petersen, C. P., Haines, B. B., Chen, J., and Sharp, P. A. (2002). Gene Silencing Using Micro-RNA Designed Hairpins. RNA 8, 842-850.


38. McManus, M. T., and Sharp, P. A. (2002). Gene Silencing in Mammals by Small Interfering RNAs. Nature Rev. Genet. 3, 737-747.


39. Miller, J. H. (1972). Experiments in Molecular Genetics (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory).


40. Mulsant, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1988). Phleomycin Resistance as a Dominant Selectable Marker in CHO Cells. Somat. Cell Mol. Genet. 14, 243-252.


41. Myslinksi, E., Ame, J. C., Krol, A., and Carbon, P. (2001). An Unusually Compact External Promoter for RNA Polymerase III Transcription of the Human H1RNA Gene. Nuc. Acids Res. 29, 2502-2509.


42. Napoli, C., Lemieux, C., and Jorgensen, R. (1990). Introduction of a Chalcone Synthase Gene into Petunia Results in Reversible Co-Suppression of Homologous Genes in trans. Plant Cell 2, 279-289.


43. Nelson, J. A., Reynolds-Kohler, C., and Smith, B. A. (1987). Negative and Positive Regulation by a Short Segment in the 5´-Flanking Region of the Human Cytomegalovirus Major Immediate-Early Gene. Molec. Cell. Biol. 7, 4125-4129.


44. Nykanen, A., Haley, B., and Zamore, P. D. (2001). ATP Requirements and Small Interfering RNA Structure in the RNA Interference Pathway. Cell 107, 309-321.


45. Paddison, P. J., Caudy, A. A., Bernstein, E., Hannon, G. J., and Conklin, D. S. (2002). Short Hairpin RNAs (shRNAs) Induce Sequence-Specific Silencing in Mammalian Cells. Genes Dev. 16, 948-958.


46. Paul, C. P., Good, P. D., Winer, I., and Engelke, D. R. (2002). Effective Expression of Small Interfering RNA in Human Cells. Nat. Biotechnol. 20, 505-508.


47. Paule, M. R., and White, R. J. (2000). Transcription by RNA Polymerases I and III. Nuc. Acids Res. 28, 1283-1298.


48. Perez, P., Tiraby, G., Kallerhoff, J., and Perret, J. (1989). Phleomycin Resistance as a Dominant Selectable Marker for Plant Cell Transformation. Plant Mol. Biol. 13, 365-373.


49. Plasterk, R. H. A., and Ketting, R. F. (2000). The Silence of the Genes. Curr. Opin. Genet. Dev. 10, 562-567.


50. Postle, K., Nguyen, T. T., and Bertrand, K. P. (1984). Nucleotide Sequence of the Repressor Gene of the Tn10 Tetracycline Resistance Determinant. Nuc. Acids Res. 12, 4849-4863.


51. Rietveld, L. E., Caldenhoven, E., and Stunnenberg, H. G. (2002). In vivo Repression of an Erythroid-Specific Gene by Distinct Corepressor Complexes. EMBO J. 21, 1389-1397.


52. Romano, N., and Macino, G. (1992). Quelling: Transient Inactivation of Gene Expression in Neurospora crassa by Transformation with Homologous Sequences. Mol. Microbiol. 6, 3343-3353.


53. Shigekawa, K., and Dower, W. J. (1988). Electroporation of Eukaryotes and Prokaryotes: A General Approach to the Introduction of Macromolecules into Cells. BioTechniques 6, 742-751.


54. Smith, C. J., Watson, C. F., Bird, C. R., Ray, J., Schuch, W., and Grierson, D. (1990). Expression of a Truncated Tomato Polygalacturonase Gene Inhibits Expression of the Endogenous Gene in Transgenic Plants. Mol. Gen. Genet. 224, 477-481.


55. Sui, G., Soohoo, C., Affar, E. B., Gay, F., Shi, Y., Forrester, W. C., and Shi, Y. (2002). A DNA Vector-Based RNAi Technology to Suppress Gene Expression in Mammalian Cells. Proc. Natl. Acad. Sci. USA 99, 5515-5520.


56. Takahashi, M., Degenkolb, J., and Hillen, W. (1991). Determination of the Equilibrium Association Constant Between Tet Repressor and Tetracycline at Limiting Mg2+ Concentrations: A Generally Applicable Method for Effector Dependent High Affinity Complexes. Anal. Biochem. 199, 197-202.


57. van der Krol, A. R., Mur, L. A., Beld, M., Mol, J. N., and Stuitje, A. R. (1990). Flavonoid Genes in Petunia: Addition of a Limited Number of Gene Copies May Lead to a Suppression of Gene Expression. Plant Cell 2, 291-299.


58. Voinnet, O., Pinto, Y. M., and Baulcombe, D. C. (1999). Suppression of Gene Silencing: A General Strategy Used by Diverse DNA and RNA Viruses of Plants. Proc. Natl. Acad. Sci. USA 96, 14147-14152.


59. Weiss, B., Jacquemin-Sablon, A., Live, T. R., Fareed, G. C., and Richardson, C. C. (1968). Enzymatic Breakage and Joining of Deoxyribonucleic Acid. VI. Further Purification and Properties of Polynucleotide Ligase from Escherichia coli Infected with Bacteriophage T4. J. Biol. Chem. 243, 4543-4555.


60. White, R. J. (1998). RNA Polymerase III Transcription (New York, NY: Springer-Verlag).


61. Wigler, M., Silverstein, S., Lee, L.-S., Pellicer, A., Cheng, Y.-C., and Axel, R. (1977). Transfer of Purified Herpes Virus Thymidine Kinase Gene to Cultured Mouse Cells. Cell 11, 223-232.


62. Yao, F., Svensjo, T., Winkler, T., Lu, M., Eriksson, C., and Eriksson, E. (1998). Tetracycline Repressor, tetR, Rather than the tetR-Mammalian Cell Transcription Factor Fusion Derivatives, Regulates Inducible Gene Expression in Mammalian Cells. Hum. Gene Ther. 9, 1939-1950.


63. Yu, J. Y., DeRuiter, S. L., and Turner, D. L. (2002). RNA Interference by Expression of Short-interfering RNAs and Hairpin RNAs in Mammalian Cells. Proc. Natl. Acad. Sci. USA 99, 6047-6052.


64. Zamore, P. D. (2001). RNA Interference: Listening to the Sound of Silence. Nat. Struct. Biol. 8, 746-750.