TECHNICAL MANUAL

Dharmacon™
shMIMIC Inducible
Lentiviral microRNA

Contents
1 Lentiviral Particle Product Safety Level Information........................................................................................3
2 Introduction to shMIMIC Inducible microRNA Lentiviral Particles.................................................................4
Dissecting Endogenous RNA Interference Mechanisms..............................................................................................................4
Innovative shMIMIC Inducible microRNA Vector Design.............................................................................................................5
An Optimized Inducible Mature MicroRNA Expression in a Single Vector................................................................................7
Importance of Promoter Activity for shMIMIC microRNA Expression........................................................................................8Transduction of shMIMIC Inducible microRNA Lentiviral Particles at Recommended
Low Multiplicities of Infection (MOI).........................................................................................................................................................9
3 Protocols for Optimization of Transduction Conditions and Choosing the Optimal
SMARTchoice Inducible Vector............................................................................................................................9
Safety Precautions........................................................................................................................................................................................9
Materials Not Supplied...............................................................................................................................................................................9
Determining Cell Density and Transduction Conditions...........................................................................................................10
4 Ordering shMIMIC Inducible microRNA Lentiviral Particles with Vector-matched
Positive and Negative RNAi Controls................................................................................................................16
Placing An Order....................................................................................................................................................................................16
Recommended Experimental Controls.........................................................................................................................................16
SMARTvector Inducible Non-targeting Control......................................................................................................................... 17
SMARTvector Inducible Positive Control shRNAs...................................................................................................................... 17
5 Mature microRNA Over-expression Protocols Using shMIMIC Inducible
Lentiviral microRNAs.......................................................................................................................................... 17
Optimal Usage........................................................................................................................................................................................ 17
Determining Functional Titer in the Cells of Interest............................................................................................................... 17
Calculating the Volume of Lentiviral Particles to Use in a Transduction...........................................................................20
Optimizing Doxycycline Concentration........................................................................................................................................20
Identifying the Optimal Doxycycline Dose for the Cells of Interest....................................................................................21
Establishing Stable Cell Populations...............................................................................................................................................22
Establishing a Stable Cell Line...........................................................................................................................................................22
Inducible Gene Silencing Without Establishing Stable Cell Lines.......................................................................................23
6 Appendix..............................................................................................................................................................23
Lentiviral Packaging ................................................................................................................................................................................. 23
Lentiviral Particle Titering...................................................................................................................................................................... 23
Stability & Storage.......................................................................................................................................................................................24
Quality Assurance and Control.............................................................................................................................................................24
Trans-Lentiviral Packaging System Biosafety Features...............................................................................................................24
7 Frequently Asked Questions..............................................................................................................................25
8 References............................................................................................................................................................26
9 Limited Use Licenses...........................................................................................................................................26

shMIMIC Inducible Lentiviral microRNA
Technical Manual

1

Lentiviral particle product safety level information
This Lentiviral particle product safety level information constitutes product documentation according to clause 1of the product terms and conditions. It is applicable to the following lentiviral particle products:•

Dharmacon™ SMARTvector™ shRNAs•

Dharmacon™ SMARTvector™ Promoter Selection Plate•

Dharmacon™ SMARTvector™ Positive and Negative RNAi Controls•

Dharmacon™ SMARTvector™ Inducible shRNAs•

Dharmacon™ SMARTvector™ shMIMIC™ microRNAs•

Dharmacon™ shMIMIC™ Inducible microRNAs•

Dharmacon™ GIPZ™ shRNAs•

Dharmacon™ Precision™ LentiORFs•

Dharmacon™ Decode™ Pooled RNAi Screening Libraries

The Products are solely for internal research use (as set forth in the product rerms and conditions) in laboratories where
the containment measures stated below and in applicable laws and regulations are met. Products may not be used for
diagnostic, therapeutic or other commercial purposes and may not to be administered to humans for any purpose or toanimals for therapeutic purposes. The Products are replication-incompetent, self-inactivating (SIN) and non-pathogenic(do not cause infectious human disease).
Any investigator who purchases Dharmacon lentiviral particle products is responsible for consulting with their institution’s healthand biosafety personnel for specific guidelines on the handling of lentiviral vector particles. Furthermore, each investigator is fully
responsible for obtaining the required permissions for research using and the acceptance of replication-incompetent SIN lentiviral
vectors and replication-defective lentiviral particles into their local jurisdiction and institution.

For questions concerning the design or production of the products, please contact our Technical Support team.
Dharmacon, Inc.
2650 Crescent Drive
Lafayette, CO 80026 USA
Tel: 1.800.235.9880; 303.604.9499
Email: ts.dharmacon@horizondiscovery.com
In the US:
For US guidance on containment for lentiviral
vectors, please refer to:
1. The Recombinant DNA Advisory
Committee (RAC) guidelines for research withlentiviral vectors (https://osp.od.nih.gov/
wp-content/uploads/Lenti_Containment_
Guidance.pdf);
2. The U.S. Department of Health and Human
Services Centers for Disease Control andPrevention and National Institutes of
Health, Biosafety in Microbiological andBiomedical Laboratories (BMBL);

3. The NIH Guidelines For Research Involving
Recombinant DNA Molecules (NIH Guidelines), (https://osp.od.nih.gov/wp-content/
uploads/2013/06/NIH_Guidelines.pdf).
In the EU:
For the EU directives, please consult the following:
1. Council Directive 2009/41/EC of theEuropean Parliament and of the Councilof 6 May 2009 on the contained use ofgenetically modified micro-organisms.
(revised version of Directive 90/219/EEC of theEuropean Parliament and of the Council of 23
April 1990 on the contained use of geneticallymodified micro-organisms, amended byCouncil Directive 98/81/EC of
26 October 1998); and2. Council Directive 2001/18/EC of the European
Parliament and of the Council of 12 March on
the deliberate release into the environment ofgenetically modified organisms and repealing
Council Directive 90/220/EEC.

In Germany:
Required Containment Measures: The containment requirements as stated in the German
Genetic Safety Ordinance (GentechnikSicherheitsverordnung) of Safety Level 2* or higher
have been assigned to the handling of theabove-mentioned lentiviral vector particles.
Please note a higher Security Level might berequired if the lentiviral vector particles are used
for genetic engineering operations with otherproducts which require a higher Security Level.
*Safety Level 2: activities of low risk for humanhealth and the environment by the state ofscientific knowledge (Stand der Wissenschaft).
For the German regulations, please consult
the following:
1. German Genetic Engineering Act(Gentechnikgesetz – GenTG); and2. Genetic Engineering Safety Ordinance(Gentechnik-Sicherheitsverordnung – GenTSV).

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shMIMIC Inducible Lentiviral microRNA
Technical Manual2

Introduction to shMIMIC inducible microRNA lentiviral particles
Dissecting endogenous RNA interference mechanisms: microRNA roles in fundamental biology
microRNAs are endogenous small (primarily 21- to 23-nucleotides) noncoding RNAs that are highly conserved acrossboth mammalian and non-mammalian species. These regulatory RNAs differentially modulate the spatial and temporal
expression of messenger RNAs (mRNAs) across the genome and have been estimated to regulate roughly 50% of all
mRNAs in vertebrates (Friedman et al., 2009; Bartel 2009).

microRNA Pathway
Nucleuspri-microRNA
Microprocessor(Drosha/DGCR8)
pre-microRNA
Exportin-5

Cytoplasm

Dicer complex

Mature microRNA(1)

Figure 1. The endogenous mammalian microRNA pathway startingwith transcription of the microRNA gene in the nucleus and ending
with two options for gene down-regulation: (1) seed-mediatedmRNA degradation or translational repression and (2) catalyticmRNA cleavage mediated by full sequence complementarity.

RISC

(2)

mRNA degradation/
Catalytic mRNAtranslational repression
cleavage

Since the discovery of the first microRNA gene, lin-4 (Lee et al., 1993; Wightman et al., 1993), thousands of microRNA
genes have been identified, representing up to 10% of the genome. An increasing number of investigations reveal
a breadth and depth of gene modulation that underscores the importance of these small RNAs.
microRNA biogenesis in mammals begins with the transcription of a microRNA gene that produces a capped andpolyadenylated primary microRNA (pri-microRNA) containing local hairpin structures with imperfect base-pairing
through the stems (Figure 1). Processing by the Drosha-DGCR8 complex results in a ~ 75 nucleotide stem-loop molecule(the precursor, or pre-microRNA), which is then shuttled out of the nucleus into the cytoplasm (Kim 2005; Kim, Han
et al., 2009). Subsequent digestion of the pre-microRNA by the endonuclease Dicer yields a transient, double-stranded
microRNA. To modulate gene expression, the double-stranded microRNA unwinds, and the resulting single-stranded
mature microRNA associates with a protein complex to form the RNA Induced Silencing Complex (RISC). In this context,
the microRNA imperfectly base-pairs primarily with sequences in the 3' untranslated regions (3' UTRs) of target mRNAs
and modulates gene expression through transcript destabilization or translational attenuation.
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shMIMIC Inducible Lentiviral microRNA
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While the list of newly discovered microRNAs continues to grow, the function and gene targets of many microRNAs
remain unknown. To address this, researchers utilize synthetic microRNA mimics and inhibitors (such as the Dharmacon
miRIDIAN Mimics and Hairpin Inhibitors) to unravel the contribution that individual microRNAs make to both normal
and diseased cellular physiology. While synthetic mimics and inhibitors can accurately simulate microRNA gain- andloss-of-function, respectively, and thereby facilitate a greater understanding of microRNA function, in some cases thecell type or the assay being employed is incompatible with use of synthetic molecules. For example, the cells being
used in a particular study can be refractory to the principal method of introducing synthetic microRNA mimics into cells,such as, lipid-mediated transfection. In other instances, the assay requires a period of time that extends beyond theeffective functional window provided by synthetic molecules. For these reasons, alternative methods for delivering andmaintaining expression of microRNA mimics are necessary.
Viral delivery systems provide a particularly effective approach for delivering genetic information into cells. While
several viral vector systems have been developed, those based on pseudotyped lentiviral vectors have proven to
be among the most versatile, by providing 1) delivery of large genomic payloads, 2) broad tropism, and 3) stable
expression by integration of the lentiviral payload into the host genome. Lentiviral vectors are unique in their ability toefficiently deliver genetic payload into both dividing and non-dividing cells. Thus, lentiviral platforms are well-suited forexperiments in stem cells and cells derived from primary tissues, neuronal and hematopoietic lineages. Our scientists
have combined expertise in microRNA biology and lentiviral vector development to create a novel, proprietary microRNA
expression tool. The shMIMIC Inducible Lentiviral microRNA is an ideal microRNA research tool for robust over-expressionof mature microRNAs.

Innovative shMIMIC inducible microRNA vector design
The shMIMIC Lentiviral microRNA design is based on a series of systematic experiments carried out during thedevelopment of the SMARTvector RNAi platform. The Inducible shMIMIC Lentiviral microRNA vectors combined theadvantages of the SMARTchoice platform with those of the state-of-the-art Tet-On® 3G induction system to develop atightly regulated, single-vector, inducible mature microRNA expression system. Figure 2 illustrates the general process
by which lentiviral-based shMIMC microRNAs enter the cell and integrate the genomic payload, including the microRNA
expression cassette, into the host cell genome. With each cellular division, the integrated sequences are replicated andpassed on to daughter cells, thus allowing controlled expression of the mature microRNA sequence throughout
the population.
Lentiviral particle carryingshMIMIC lentiviral vector

mature microRNA
duplex
Dicer

proteinexpression

mRNA
RNA

DNA

primary microRNA
expression

Figure 2. The shMIMIC system utilizes lentiviral vectors
to stably deliver and express gene down-regulating
triggers capable of entering the RNAi pathway.

ion
at
gr
e
t
in

reversetranscription

preintegration
complex
nucleus5

shMIMIC Inducible Lentiviral microRNA
Technical Manual
shMIMIC Inducible microRNA vectors (Figure 3) possess the following enhanced features for robust, regulated andreproducible mature microRNA expression:
1.

Optimized universal microRNA scaffold: microRNAs are the endogenous substrates of the RNAi pathway.
Transcribed in the nucleus as stem-loop structures containing imperfect basepairing in the stem,
these noncoding RNAs are processed by multiple elements of the RNAi pathway to generate short ~16-28 base-pair duplexes capable of associating with the RNA Induced Silencing Complex (RISC) to target
genes for down-regulation. Our scientists screened a panel of human microRNAs and selected an efficiently
processed primary microRNA for the universal scaffold.

2. Rational design for over-expression of each mature human, mouse and rat microRNA in the miRBase
database (www.mirbase.org): Each mature microRNA is systematically embedded within the universal scaffold
and cloned into the lentiviral vector backbone. The use of a universal primary context ensures that each mature
microRNA is correctly and consistently processed in the cell, thus minimizing sequence variability that can confoundinterpretation of microRNA-induced phenotypes.
3. Tightly regulated, inducible shMIMIC microRNA expression: shMIMIC Inducible microRNAs combine all of theadvantages of the SMARTvector platform with those of the recently developed Tet-On® 3G tetracycline-inducible
expression system. Our unique, single-vector delivery system permits the rapid development of cells with tightly
controlled mature microRNA expression. The minimal leakiness and potent activation of the Tet-On® 3G promoter,
an RNA polymerase II promoter, ensures near wild-type expression of the mature microRNA in the absence of
doxycycline and robust over-expression upon exposure to doxycycline. This regulated mature microRNA overexpression offers additional experimental control, providing numerous experimental advantages, including
regulated over-expression of mature microRNAs that result in cell death.
4. TurboGFP™ or TurboRFP™ reporter genes: The shMIMIC Inducible microRNA platform includes either a TurboGFPor TurboRFP (Evrogen, Moscow, Russia) reporter gene to facilitate assessment and optimization of experimental
transduction conditions. Because the mRNA encoding the fluorescent reporter is directly linked to the shMIMIC
microRNA, expression of TurboGFP or TurboRFP allows for visual tracking of mature microRNA expression. Expressionof TurboGFP or TurboRFP occurs only in the presence of doxycycline and fluorescence indicates that the expressionof the mature microRNA has been induced.
5. Mammalian drug-selection marker: The puromycin resistance (PuroR) gene is a feature of all SMARTchoicelentiviral constructs and enables rapid selection of stable cell lines. In the shMIMIC Inducible microRNA vector, PuroR
is expressed under the control of one of four constitutive promoter options, thus permitting selection of transduced
cells independently of mature microRNA expression.
6. SMARTchoice RNA Pol II promoter options: The activity of RNA polymerase II promoters can vary in different
cellular contexts. The shMIMIC Inducible platform provides a choice of four robust, well-characterized
RNA polymerase II promoters (mCMV, PGK, mEF1α and mEF1α) to control the expression of PuroR and the
doxycycline-dependent transactivator, Tet-On® 3G. This ensures that a functional inducible shMIMIC microRNA vector
can be utilized in virtually any mammalian cell type of interest.

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shMIMIC Inducible Lentiviral microRNA
Technical Manual

mCMV
PGK
mEF1α
hEF1α

SMARTchoicepromoters

PTRE3G

5' LTR

Ψ

RRE

WPRE

3' SIN LTR

or

tRFP

Figure 3. Elements of the shMIMIC
Inducible Lentiviral microRNA vector.

PuroR 2a Tet-On 3G

tGFP
SMARTvector
universal scaffold

Vector element

Utility

5' LTR

5' Long Terminal Repeat is necessary for lentiviral particle production and integration of the construct into the host cell genomeΨPsi packaging sequence allows lentiviral genome packaging using lentiviral packaging systems

RRE

Rev Response Element enhances titer by increasing packaging efficiency of full-length lentiviral genomes

PTRE3G

Inducible promoter with Tetracycline Response Elements which is activated by the Tet-On 3G protein in the presence of doxycyclinetGFP or tRFP

TurboGFP or TurboRFP reporter for visual tracking of transduction and expression upon doxycycline induction

SMARTvector universal scaffold

Optimized patented scaffold based on native primary microRNA in which mature microRNA sequence is embedded

Puro

Puromycin resistance gene permits antibiotic selection of transduced cellsR

2aSelf-cleaving peptide that enables the expression of both PuroR and Tet-On 3G transactivator from a single RNA pol II promoter

Tet-On 3G

Encodes the doxycycline-regulated transactivator protein, which binds to PTRE3G only in the presence of doxycycline

WPRE

Woodchuck Hepatitis Post-transcriptional Regulatory Element enhances transgene expression in target cells

3' SIN LTR

3' Self-inactivating Long Terminal Repeat for generation of replication-incompetent lentiviral particles

An optimized inducible mature microRNA expression in a single vector
The shMIMIC Inducible microRNA vectors utilize the Tet-On® 3G bipartite induction system. This tightly regulated system
consists of an inducible RNA polymerase II promoter, which has been optimized for both minimal basal expression
and potent activation upon induction (Loew, 2010). In the presence of doxycycline, the TRE3G promoter is bound andactivated by the constitutively expressed Tet-On® 3G transactivator protein (Figure 4), which is also encoded within theshMIMIC Inducible Lentiviral microRNA vector. This protein was the product of an in vitro evolution and selection processthat resulted in significantly higher sensitivity to doxycycline than the original Tet-On® 3G protein (also known as rtTA).
Together, Tet-On® 3G protein and TRE3G promoter permit tight regulation of the mature microRNA, including potent
induction, even at the low doxycycline doses that are required in vivo.

Tet-On® 3GProtein

Doxycycline

No Doxycycline

X OFF

Plus Doxycycline
ON

Figure 4. Tet-On® 3G transactivator protein only binds the
TRE3G promoter sequence when doxycycline is present,
which in turn activates transcription.

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shMIMIC Inducible Lentiviral microRNA
Technical Manual

Importance of promoter activity for shMIMIC microRNA expression
In order for shMIMIC Inducible microRNAs to function in a broad range of cells, the constitutive promoter must be
active enough to produce sufficient quantities of PuroR and Tet-On® 3G transactivator protein to permit both selectionof transduced cells and inducible expression of the mature microRNA. Depending on the specific cell type, promoter
activity may be nonexistent, weak or silenced by epigenetic mechanisms over time, despite successful transduction
and integration. The fluorescent images shown in Figure 5 demonstrate how promoter performance varies significantly
across different cell lines which, in turn, can influence the efficacy and potency of over-expressing mature microRNAs.
Due to the proximity of the constitutive and the TRE3G promoters in these vectors, a particular constitutive promoter
does not always behave as it would when isolated in a single promoter system. This phenomenon is commonly referred
to as promoter interference (Curtin, 2008). It is recommended to test control particles for each vector, using inducible
TurboGFP expression as a measure of vector activity in your cell type of interest.

SMARTchoice Inducible vector customization options include four promoter and two reporter options. To determine
the optimal shMIMIC Inducible microRNA vector for your cell type of interest, utilize the SMARTchoice Inducible
Non-targeting Control 4-Pack to quickly identify the promoter that drives maximal shMIMIC microRNA expression inyour cells, simply by examining doxycycline-induced TurboGFP expression (see Protocol in Chapter 3).

hEF1α

mCMV

hEF1α

mCMV

hEF1α

mCMV

mEF1α

PGK

mEF1α

PGK

mEF1α

PGK

A549 (human lung adenocarcinoma)

HeLa (human cervical adenocarcinoma)

HUVEC (primary human endothelial)

hEF1α

mCMV

hEF1α

mCMV

hEF1α

mCMV

mEF1α

PGK

mEF1α

PGK

mEF1α

PGK

IMR90 (primary human fibroblast)

NIH/3T3 (mouse embryonic fibroblast)U2OS (human osteosarcoma)

Figure 5. shMIMIC Inducible microRNA promoter activities differ across cell types. Inducible Non-targeting Control lentiviral particles were used to transduce theindicated cell types at MOI = 0.3; 24 hours after transduction, expression of the non-targeting shRNA and TurboGFP was induced with 1 µg/mL doxycycline. After 48 hoursof culture in the presence of doxycycline, cells were stained with Hoescht-33342 and nuclei (blue) and TurboGFP (green) were imaged. While ~ 30% of the cells in the field
have been transduced, some images may appear to contain fewer than 30% TurboGFP-positive cells due to low TurboGFP expression, indicative of low constitutive promoter
activity, in a particular cell type.

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shMIMIC Inducible Lentiviral microRNA
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Transduction of shMIMIC inducible microRNA lentiviral at recommended low multiplicities ofinfection (MOI)The Tet-On® 3G system has been optimized to minimize leaky activity. However, all inducible expression systems display
some level of basal or “leaky” activity in the absence of the inducer. The level of basal expression increases as the copy
number of shMIMIC microRNA increases with the number of proviral genomes integrated (for example with increasing
the multiplicity of infection, MOI). Additionally, toxicity of Tet-On proteins has been reported (Sisson, 2006; Whitsett,2006; Morimoto, 2009), and the expression level of Tet-On® 3G is directly correlated with MOI. Therefore, in most
instances, a single integration of proviral genome per cell provides the optimal balance between minimized leakiness,
maximized inducible mature microRNA expression, and minimal non-specific effects resulting from expression of Tet-On®
3G protein.
Nevertheless, because shMIMIC Inducible Lentiviral microRNA are provided as concentrated lentiviral particles, MOIs ofgreater than one are often achievable. While higher MOIs may enhance phenotypic results by increasing the expressionof the mature microRNA , it is important to consider the possibility of low-level mature microRNA expression in theabsence of doxycycline induction.an 25% confluent.

3

Protocols for optimization of transduction conditions and 			choosing the optimal SMARTchoice inducible vector
Safety precautions
The following protocols describe how to determine optimal conditions tolerated by cells of interest under whichtransductions can subsequently be performed. The goal is to optimize cell density, Polybrene concentrations,presence or absence of serum and duration of transduction. It is critical to comprehensively read and understandthe recommended steps in each protocol prior to initiating the promoter selection assay. Preparation and handlingof the shMIMIC Inducible Lentiviral microRNAs and subsequent transduction plates must always be performed within aClass II/Type A2 biological safety cabinet. Any surfaces coming in contact with the lentiviral particles, including pipettetips and plastic ware, should be sanitized by bleach or Lysol™ disinfectant prior to disposal. To facilitate disposal, set up aliquid waste reservoir inside the biological safety cabinet consisting of a 1 L beaker containing 100 mL of bleach or Lysol™
disinfectant. A biohazardous waste bag should also be placed within the biological safety cabinet.

Materials not supplied•

Polybrene (American Bioanalytical Cat #AB01643)
The use of Polybrene may improve transduction efficiency of lentiviral particles in some cell types and is optional.

•

96-well tissue culture plates (Nunc™ Cat #12-565-66).

•

Deep-well 96-well plate (Nunc™ Cat #12-565-553).

•

Multichannel (8-channel) Pipette.

•

Cells of interest. See note in blue box.

•

Dulbecco’s Modified Eagle Medium (DMEM) High Glucose without L-Glut or Sodium Pyruvate(Hyclone™ Cat #SH30081.FS).

•

Doxycycline hyclate (Fisher Scientific Cat #ICN19895510).

•

alamarBlue™ Cell Viability Reagent (Thermo Scientific Cat #PI88952).
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shMIMIC Inducible Lentiviral microRNA
Technical Manual•

Preferred culture medium: the cell culture medium (including serum or supplements) recommended formaintenance and passaging of the cells of interest.

•

Base medium: the base medium (without serum or supplements) that is used to prepare the Preferredculture medium.

•Transduction medium: Base medium with or without serum and containing the appropriate concentration of
Polybrene as determined to be optimal transduction conditions for cells of interest.

•

Lysol™ disinfectant or bleach.
The passage number of cells can have a significant effect on lentiviral transduction efficiency. For this reason, cells of low passage
number (typically < 10) should be used in all experiments, and accurate records that document cell passage number should be maintained.
If you are using a recently thawed vial of cells, allow at least one passage after the thaw before performing optimization procedures.

Determining cell density and transduction conditions
Successful transduction of cells depends on a number of factors including the cell type, cell density, passage number,
MOI during transduction, purity of the lentiviral preparation and the presence and/or absence of reagents that facilitatetransduction. Additionally, your lentiviral particles are suspended in DMEM, which may not be the base medium of thepreferred culture medium for the cells of interest.
Polybrene can be toxic to certain cells. Although polybrene is not generally necessary to achieve the MOIs recommended
for shMIMIC Inducible Lentiviral microRNAs, it may aid transduction for some difficult-to-transduce cell types.
The following optimization protocol allows you to determine potential lentiviral transduction conditions that arecompatible with the cell type of interest. The protocol is designed to test multiple conditions in one experiment in a
96-well plate format as depicted below (Figure 6). This recommended protocol will test cell density, Polybrene
concentration, the presence or absence of serum and the duration of transduction (6 hours or overnight).

i. Optimization of cell density and conditions in preparation for transductions: protocol
for adherent cells
Day 1:
1.

Seed cells into two 96-well culture plates at the appropriate cell density following the template depicted in
Figure 6. Seed cells in a total volume of 100 μL of the preferred culture medium and place 96-well culture plates into
incubator for overnight culture under the appropriate conditions (temperature and CO2 concentration).

2.

Generally, transductions with lentiviral particles should be performed when cells are approximately 40% confluent.
This provides cells the space to replicate in culture. Because cells can differ significantly with respect to size andmorphology, the number of cells seeded should reflect this difference. The number of cells shown in Figure 6(5,000, 7,500, and 10,000) is specified only to depict a range of cell concentrations that should be seeded acrossthe 96-well plate. The actual number of the cells seeded for the specific cells of interest will vary depending on
the size and morphology and will depend on previous experience.
Day 2:

1.

Visually inspect each well under a microscope. Note the confluency for each of the three different cell concentrations
seeded on Day 1. If all three cell concentrations seeded resulted in > 90% confluency, then it is necessary to re-seed the cells
using a lower range of cell densities.

2.

Prepare two sets of transduction medium, one without serum and one with serum.

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shMIMIC Inducible Lentiviral microRNA
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Figure 6. Example of 96-well plate layout for the optimizationof transduction conditions. This plate layout tests three celldensities [5,000 (5K), 7,500 (7.5K) and 10,000 (10K) cells perwell], eight concentrations of Polybrene (0-14 µg/mL) andmedium with or without serum.

Polybrene (µg/mL)

Cell Density

0

A

2

B

4

C

6

D

8

E

10

F

12

G

14

H

5K

7.5K

10K

without serum0

5K

7.5K

10K

with serum

Transduction medium without serum:»» Prepare 10 mL of transduction medium containing no serum by pipetting a 1:1 mixture of DMEM to basemedium. Note that Dharmacon provides lentiviral particles in DMEM. Thus, testing a recipe for suitabletransduction medium must include DMEM.
»»

Transfer 700 μL aliquots of this mixture into eight wells of a sterile deep-well 96-well plate. Use these wells to
prepare a series of transduction medium formulations containing eight different Polybrene concentrations
ranging from 0-14 μg/mL, increasing concentration in 2 μg/mL increments (Figure 7).

Transduction medium with serum:»» Prepare a similar series of wells with transduction medium consisting of a 1:1 mixture of DMEM to basemedium and containing serum at the concentration that is appropriate for the cells of interest. For example,
if the preferred culture medium contains 10% serum, then the final concentration of the transduction medium
should also be 10%.
3.

Incubate cells with Transduction Medium.
Two sets of incubations will be performed using each of the transduction medium formulations (withand without serum): one plate for six hours (plate 1) and the other plate for an overnight incubation for
16-20 hours (plate 2).
a. Remove culture plates from incubator and place in biological safety cabinet.
b. Using a multichannel pipette, carefully aspirate preferred culture medium from each well of 96-well culture
plates being careful not to dislodge cells from bottom of well. Aspirate into a liquid waste reservoir.
c. Using a multichannel pipette, transfer 50 μL of transduction medium (with or without serum) containing
the range of Polybrene concentrations (0-14 μg/mL) to cells seeded in 96-well culture plate. Transfer transduction
medium in format depicted in Figure 7.
d. Return culture plates to incubator and culture under the appropriate conditions.
e. After 6 hours, add 100 μL of preferred culture medium directly to each well of plate 1 and return to incubatorfor overnight culturing. At this time, do not remove the transduction medium from the well prior to adding thepreferred culture medium. To adjust for the absence of serum (transductions without serum only) make up the
difference by including additional serum to the preferred culture medium. For example, if your cells require
10% serum, then increase the serum concentration of the preferred culture medium to 15%. Adding 100 μL ofthis culture medium to the 50 μL of transduction.
11

shMIMIC Inducible Lentiviral microRNA
Technical Manual• If Polybrene is toxic to the cells, DEAE-Dextran (1-10 µg/mL) may be substituted.
• If no-serum conditions affect cellular viability then optimization can be performed using a lower serum concentration (1-3%) and thesame plate format as in Figure 7.

rum

se
out

Figure 7. Example of plate layout for preparation ofTransduction Medium. Transduction Medium with (bluewells) or without (purple wells) serum containing a rangeof Polybrene concentrations in a deep-well, 96-well plate.
White wells are empty.

Polybrene (µg/mL)

h
wit
0

A

2

B

4

C

6

D

8

E

10

F

12

G

14

H

m

eru

hs
wit

Deep-well plate

Day 3:
After overnight incubation with Transduction Medium (16-20 hours), add 100 µL of Preferred Culture Medium directly
to each well of plate 2. Adjust serum concentration as described above to the wells without serum.
Day 4:
Using a microscope, examine all wells of 96-well plates. Record the confluency and any morphological or phenotypicalterations that may be present.
Day 5:
Examine cultures for cell morphology or presence of phenotypic changes. Cell viability should be determined using
any commercially available kit, such as alamarBlue Cell Viability Reagent (Thermo Scientific).
Select the initial cell density seeded that resulted in approximately 40% confluency on Day 2. If the cells of interest cantolerate serum-free conditions then exclude serum during transduction. Note the highest concentration of Polybrene withacceptable viability for the cells of interest. Based on observations from a variety of cell lines and cell types, the lowest
concentration of serum generally results in the highest transduction efficiency. If, in subsequent protocols, it
is found that transduction efficiency is low, Transduction Media may be supplemented with this concentration of
Polybrene to enhance efficiency. See notes above in blue box.
Furthermore, if there is no evidence of significant cellular toxicity with overnight incubation (16-20 hours), then thisincubation time may be used for subsequent transductions.

ii. Optimization of cell density and conditions in preparation for transductions: protocol
for suspension cells
Non-adherent or suspension cells should be counted and plated at the time of transduction and notincubated overnight.
12

shMIMIC Inducible Lentiviral microRNA
Technical ManualDay 1:
1.

Determine the number of suspension cells per mL. Transfer enough cells to seed two 96-well culture plates into
two sterile centrifuge tubes (include additional 20% volume of cells to ensure adequate volumes for transfer).
Centrifuge cells at low speed to pellet cells. Pour off supernatant containing old culture medium. Tap bottom ofcentrifuge tube gently to dislodge cell pellets. Resuspend cells in one centrifuge tube with Base Medium containing
no serum and cells in the other centrifuge tube in Base Medium containing appropriate concentration of serum
for cells of interest. Resuspend in enough volume so that the appropriate number of cells are seeded in a total
volume of 25 μL per well.

2.

Transduction Medium formulations (with and without serum; 0-14 μg/mL Polybrene concentration) should be
prepared essentially as described above. However, because Transduction Medium will be added to cells resuspended
in 25 μL of Base Medium (with and without serum), Polybrene and serum concentrations of Transduction Medium
will need to be adjusted accordingly.
Transduction medium for suspension cells:»» Prepare 10 mL of Transduction Medium without serum by pipetting a 1:1 mixture of DMEM to Base Medium.
Transfer 700 μL aliquots of this mixture into eight wells of a sterile deep-well 96-well plate. Use these wells to
prepare a series of Transduction Medium formulations containing eight different Polybrene concentrations suchthat the final concentration will range from 0-14 μg/mL in 2 μg/mL increments (as depicted in Figure 6). Polybrene
concentration is increased to account for the additional 25 μL of Base Medium, which does not already contain
Polybrene. For example, well 2 contains 3 μg/mL Polybrene; adding 50 μL of Transduction Medium with a
Polybrene concentration of 3 μg/mL to 25 μL of Base Medium containing no Polybrene will result in a final
concentration of 2 μg/mL.
»»

Use Table 1 to prepare the eight Transduction Medium formulations. Two sets of eight wells of Transduction
Medium should be prepared as before (Figure 7), one set without serum and one with serum.
Table 1. Transduction Medium formulations for preparing media with eight different Polybrene concentrations.

Polybrene concentration oftransduction medium

Final Polybrene concentration after
adding to suspension cells

Well 1none

0 µg/mL

Well 23 µg/mL

2 µg/mL

Well 36 µg/mL

4 µg/mL

Well 49 µg/mL

6 µg/mL

Well 512 µg/mL

8 µg/mL

Well 615 µg/mL

10 µg/mL

Well 718 µg/mL

12 µg/mL

Well 821 µg/mL

14 µg/mL

3.

Incubate cells with Transduction Medium.
Two sets of incubations will be performed using each Transduction Medium formulation (with and without serum):
one plate for 6 hour incubation (plate 1) and the other plate for 16-20 hour overnight incubation (plate 2). Using a
multichannel pipette, transfer 50 μL of Transduction Medium to appropriate wells and place 96-well culture plates
into incubator under appropriate conditions (temperature and CO2 concentration).

4.

After 6 hours, add 75 μL of Preferred Culture Medium directly to each well in plate 1 and return to incubator forovernight culturing. Adjust serum concentration in wells containing no serum. For example, if cells require
10% serum, increase the serum concentration of Preferred Culture Medium to 20%. Adding 75 μL of this culture
medium to the 75 μL of Transduction Medium in the well will result in a final serum concentration of 10%.

Day 2:
After overnight incubation with Transduction Medium (16-20 hours), add 75 μL of Preferred Culture Medium directly
to each well in plate 2. Adjust serum concentration as described above to wells containing no serum.
13

shMIMIC Inducible Lentiviral microRNA
Technical ManualDay 3:
Using a microscope, examine all wells of 96-well plate and note any morphological or phenotypic alterations present.
Day 4-5:
Examine cultures for cell morphology or presence of phenotypic changes. Cell viability should be determined using
any commercially available kit, such as alamarBlue™ Cell Viability Assay Reagent.
Select the appropriate density (for example, ~ 40% confluency for the specific cells of interest) on Day 2. If the cells ofinterest can tolerate serum-free conditions then exclude serum during transduction. Note the highest concentrationof Polybrene with acceptable viability for the cells of interest. Based on observations from a variety of cell lines andcell types, the lowest concentration of serum generally results in the highest transduction efficiency. If, in subsequent
protocols, it is found that transduction efficiency is low, Transduction Medium may be supplemented with thisconcentration of Polybrene to enhance efficiency. See notes below in blue box.
• If Polybrene is toxic to the cells, DEAE-Dextran (1-10 µg/mL) may be substituted.
• If no-serum conditions affect cellular viability then optimization can be performed using a lower serum concentration (1-3%) and thesame plate format as in Figure 7.

Furthermore, if there is no evidence of significant cellular toxicity with overnight incubation (16- 20 hours), then thisincubation time may be used for subsequent transductions.

iii. Using the SMARTchoice inducible non-targeting control 4-pack to identify the optimal
vector for a given cell type
SMARTchoice Inducible vector configuration options include four RNA pol II promoter options, mouse CMV, PGK, human
EF1α and mouse EF1α, all of which control the expression of puromycin resistance and the Tet-On 3G transactivator.
However, the activity of these promoters may not be known for a particular cell of interest prior to RNAi experimentation.
By utilizing the SMARTchoice Inducible Non-targeting Control 4-Pack (Cat #VSC6847), the researcher can simultaneously
evaluate which of these promoters are most active in the cells of intended study, and maximize the opportunity forexperimental success. Following the optimization of basic transduction conditions (see Protocol i. or ii.), cells aretransduced with the SMARTchoice Inducible Non-targeting Control 4-Pack lentiviral particles under the appropriate
conditions. TurboGFP expression is induced with 1 μg/mL doxycycline and the activity of each vector configuration
is observed as relative intensity of TurboGFP fluorescence. The optimal vector configuration is the one producing thehighest level of fluorescence intensity. Once the most active promoter option is qualitatively determined for the specificcells of interest, shMIMIC Inducible microRNA Lentiviral Particles or glycerol stocks expressing microRNA of interest andexperimental controls incorporating the optimal promoter and fluorescent reporter (TurboGFP or TurboRFP) of choice
can be ordered.
Day 1:
Plate cells in a 96-well culture plate at optimal cell density in appropriate growth medium as determined in previous
experiments (see Protocol i. and ii.). If working with suspension cell lines, plate cells the day of transduction.
Day 2:
1.

Prepare 10 mL of pre-warmed Transduction Medium (using serum concentrations as determined previously).

2.

Prepare the lentiviral particle dilution plate in which each column represents one of the four SMARTchoice Inducible
Non-targeting Control vector configurations (mCMV, PGK, hEF1α and mEF1α); see Figure 8 for an illustration of therecommended plate layout. To the first well at the top of each of four columns (Row A), add 90 μL of Transduction
Medium. Then add 80 μL of Transduction Medium to each well in Rows B-H, columns 1-4. Place this plate ofTransduction Medium in the incubator until Step #4.

3.

Remove your SMARTchoice Inducible Non-targeting Control 4-Pack from the -80 °C freezer and thaw the lentiviralparticles while keeping the tubes on ice. Normalize the titers of all four Non-targeting controls to the one withthe lowest titer using DMEM.

14shMIMIC Inducible Lentiviral microRNA
Technical Manual

Volume ofTransduction
Medium:Figure 8. Lentiviral particle dilution platelayout for assessing vector activity in cellsof interest using the SMARTvector Inducible
Non-targeting Control by creating a 5-fold serial
dilution. There should be no lentiviral particles
in Row H wells, only medium.

Add 10 μL of lentiviral particles
with normalized titer

F1 1MV
n E se EF
eC
a
s
u
u
m
Mo PGK Hu Mo

1

2

3

4

5

6

7

8

9

10 11 12

90 μL

A

80 μL

B

Transfer 20 μL

80 μL

C

Transfer 20 μL

80 μL

D

Transfer 20 μL

80 μL

E

Transfer 20 μL

80 μL

F

Transfer 20 μL

80 μL

G

Transfer 20 μL

80 μL

H

4.

To your dilution plate containing transduction medium, add 10 μL of each thawed, titer-normalized Inducible
Non-targeting control lentiviral particles to separate wells at the top of each column (Row A). Mix the particles
by gently pipetting up and down 3-5 times.

5.

With a multichannel pipette, transfer 20 μL of transduction medium with lentiviral particles from row A to row B.
Mix at each transfer step by gently pipetting up and down 3-5 times.

6.

Repeat the transfer of 20 μL of transduction medium with lentiviral particles from each row to the row below it
down to Row G (Figure 8), thus creating 5-fold serial dilutions for each Inducible Non-targeting control vector
configuration. Wells in row H should contain no lentiviral particles.

7.

Remove cell culture plates from the incubator and replace cell culture medium with 25 μL of transduction
medium with lentiviral particles from the corresponding well of the lentiviral particle dilution plate.

8.

Return cell culture plate with transduction medium and lentiviral particles to the incubator.

9.

After 6-20 hours (as determined in protocols i. or ii.), add 100 μL of preferred culture medium directly to eachwell without removing medium. Make the necessary adjustments to serum concentration so that the final
serum concentration matches preferred culture medium after adding to wells.

CMYK
50
100
15
10

Doxycycline is unstable in solution. It is recommended that a fresh working solution be stored for no more than three days at 4 °C.
Additionally, when inducing cells with doxycycline, the medium should be replaced with fresh medium containing fresh doxycycline
every 24-48 hours.

Day 3:
1.

After 24 hours, replace medium with preferred culture medium supplemented with 1 µg/mL of freshly
dissolved doxycycline.

2.

Return plate to incubator and culture for an additional 48 hours.

Day 5:
Examine each well of the 96-well culture plates, and for each vector, identify the row in which approximately 10-30%
of the cells are positive for TurboGFP. The majority of cells in this row carry a single integration of the viral genome.
See note in blue box below (page 16). Assess TurboGFP fluorescence intensity by FACS or microscopy using theappropriate filters (Table 2).

15

shMIMIC Inducible Lentiviral microRNA
Technical Manual

The optimal vector for your cell type is the one producing the highest level of fluorescence intensity.
Table 2. Excitation and emission maxima for TurboGFP and TurboRFP fluorescent reporters.

Fluorescent reporter

Excitation wavelength

Emission wavelength

TurboGFP

482 nm502 nmTurboRFP

553 nm574 nmTurboGFP and TurboRFP expression from a single lentiviral integration can be difficult to see without a high-quality fluorescencemicroscope. Additionally, phenol red absorbs at both the excitation and emission wavelengths of both of fluorophores. To enhancethe visibility of fluorescing cells, replace the culture medium with a medium that is low in phenol red.

iv. Determining optimal puromycin selection conditions
Day 1:
Using the same cell type and cell densities to be used in subsequent transduction procedures, plate cells
and culture overnight.
Day 2:
Replace complete growth medium with growth medium supplemented with a range of puromycin
concentrations (0.1-10 μg/mL).
Day 4:
Replace medium with fresh medium supplemented with the appropriate concentration of puromycin. Examine
cells daily and identify the minimum concentration of puromycin that efficiently kills all cells between 2-4 days following
addition of puromycin.

4

Ordering shMIMIC inducible microRNA lentiviral particles or 		
glycerol stocks with vector-matched positive and negative
RNAi controls
Placing an ordershMIMIC Inducible microRNA lentiviral particles and glycerol stocks can be ordered at dharmacon.horizondiscovery.com/
rnai/microrna/#all with one of four well-characterized promoters and either TurboGFP or TurboRFP as the fluorescentreporter (Figure 3). Order individual lentiviral constructs at 100 μL or 200 μL volumes of≥ 1 × 107 TU/mL lentiviral particles, or glycerol stocks. It is imperative to select matching positive and negative controls
with the same promoter and reporter options selected for the mature microRNA of interest.

Recommended experimental controls
Inducible negative and positive RNAi controls are cost-effective reagents for experimental optimization such asdetermining optimal conditions for transduction and doxycycline induction, prior to transductions with specific shMIMIC
Inducible miRNA lentiviral particles. Including both positive and negative controls in all experiments assures rigorous,
high-confidence data, and is required for accurate data interpretation.
We strongly recommended that SMARTvector Inducible Negative and Positive RNAi Controls be ordered in addition tolentiviral particles targeting the gene of interest. Vector-matched controls are available with options for all four promotersand two fluorescent reporters.
•••

SMARTvector Inducible Non-targeting Control
SMARTvector Inducible GAPDH Positive Control shRNA (human, mouse and rat)
SMARTvector Inducible PPIB Positive Control shRNA (human, mouse and rat)16

shMIMIC Inducible Lentiviral microRNA
Technical Manual

SMARTvector inducible non-targeting control
SMARTvector Inducible negative controls for RNAi experiments, provided as packaged lentiviral particles at≥ 1 × 107 TU/mL, or glycerol stocks are designed so that no known gene in human, mouse or rat will be targeted, thus
providing a baseline to which microRNA-specific effects can be normalized. Negative controls can also be used to assesstransduction efficiency and the effects of transduction on the cells of interest. The SMARTvector Inducible
Non-targeting Controls are available with all four SMARTchoice Inducible promoter options and both fluorescentreporters so that RNAi experiments can be performed utilizing the appropriate controls regardless of the promoter
chosen for gene-specific constructs.
Although little or no shMIMIC microRNA expression occurs in the absence of doxycycline, it is strongly recommended that avector-matched non-targeting control is used to control for potential cellular phenotypes that may be associated with doxycycline
exposure and/or expression of the fluorescent reporter.

SMARTvector inducible positive control shRNAs
SMARTvector Inducible oositive control shRNAs are provided as packaged lentiviral particles at ≥ 1 × 107 TU/mL or
glycerol stocks. The inducible shRNA positive controls permit confirmation of transduction efficiency and geneknockdown through consistent, potent silencing of non-essential, abundantly expressed reference genes. The inducible
GAPDH positive control shRNA is validated to silence glyceraldehyde-3-phosphate dehydrogenase, while the inducible
PPIB positive control shRNA is validated to target peptidylprolyl isomerase B (also known as cyclophilin B). Individual
positive control shRNAs targeting GAPDH and PPIB in human, mouse and rat models are available in each SMARTvector
Inducible vector promoter and reporter options.

5

Mature microRNA over-expression protocols using shMIMIC 		
inducible lentiviral microRNAs
Optimal usage of shMIMIC Inducible microRNA lentiviral particles
In most instances, we recommend relying on single-copy lentiviral genome integration for optimal mature microRNA
expression with minimal leakiness (Chapter 2). However, because shMIMIC Inducible microRNAs are provided asconcentrated lentiviral particles, MOIs of greater than one are often achievable if required for a specific application. Whilehigher MOIs may enhance over-expression of the mature microRNA, it is important to consider the possibility of low-level
microRNA expression in the absence of doxycycline induction.
The following protocols are designed to rapidly develop cells carrying a single copy of a shMIMIC Inducible microRNA
proviral genome, and to induce shMIMIC microRNA expression with an optimized doxycycline dosage. In general, to
produce populations of cells with a uniform MOI = 1, the cells are first transduced with enough lentiviral particles to
produce an MOI between 0.1 and 0.3. Following the lentiviral transduction, cells that were not transduced are removed
from the population through puromycin selection. The majority of cells that remain after selection will carry a single copyof the inducible shMIMIC microRNA.

Determining functional titer in the cells of interest
The functional titer of SMARTvector Lentiviral shRNA constructs in HEK293T cells is reported on the Certificate of Analysis (C of A).
The relative transduction efficiency of your cell type will likely be lower than that of HEK293T cells. The protocol below can be used
to determine your cell-line specific functional titer.

Functional titer can be determined either by counting GFP-positive colonies using fluorescence microscopy or by FACS
analysis of fluorescent shRNA constructs, or resazurin for non-fluorescent shRNA constructs. The following protocol
describes how to estimate functional titer by using non-silencing control lentiviral particles and determining titer byfluorescence microscopy.
17

shMIMIC Inducible Lentiviral microRNA
Technical Manual
Calculating the volume of lentiviral particles needed for a given MOI
Calculate the total number of transducing units (TU) that would be added to a well for a given MOI with the following equation:
TU = (MOI × CN)/VT
where, MOI = the desired MOI in the well (units are TU/cell); CN = number of cells in the well; VT = Viral Titer (units are TU/μL).
For example, if the experiment requires per well:• MOI of 0.3• Cell density of 10,000 cells per well at time of transduction; and• Lentiviral titer as functionally determined for your cell type and conditions is 1 × 107 TU/mL (= 1 × 104 TU/μL)
Then, total TUs per well is equal to:
Total TU = [(0.3 TU/cell) × (10,000 cells/well)] / 1 × 104 TU/μL = 0.3 μL of lentiviral stock/well.
Therefore, the volume of lentiviral particles with a titer of 1 × 107 TU/mL required for an MOI of 0.3 is 0.3 μL per well. It is recommended
to include biological triplicates. When making lentiviral dilutions, be certain to scale the dilution series such that volumes are sufficientenough to permit accurate pipetting well within the working range of your pipettor.

1.

The day before transduction, seed a 96-well cell culture plate (Destination Plate) with your cells at the density
determined during transduction optimization. Grow cells overnight.

2.

Make dilution medium using serum and polybrene conditions determined during transduction optimization. Make
dilutions of inducible non-silencing control lentiviral particles in a round-bottom 96-well plate (Dilution Plate). As
shown in Figure 9 and Table 3, use one row of the plate for each replicate of the dilution series of the lentiviral stock.
We recommend performing two replicates. The procedure for dilution of the lentiviral stock is described below andresults in a series of five-fold dilutions to reach a final dilution of 390,625-fold.
a.

Add 40 µL of dilution medium to wells A1 and B1. Add 80 µL of dilution medium to each well A2-A8 and B2-B8.

b.

Thaw non-silencing control lentiviral particles on ice and then add 10 µL each to wells A1 and B1. Mix contents ofeach well by pipetting up and down 10-15 times. Discard pipette tip.

c.

Transfer 20 µL from wells A1 and B1 to the corresponding wells in column 2. Mix contents of each well by
pipetting up and down 10-15 times. Discard pipette tip.

d.

Repeat transfer of 20 µL for columns 2 through 8, mixing 10-15 times for each dilution.

e.

Allow lentiviral-Polybrene complexes to form for 3-5 minutes at room temperature ( only if using Polybrene).

3.

Remove culture medium from the cells in the 96-well plate Destination Plate

4.

Transfer 25 µL of each dilution of lentiviral particles from the Dilution Plate to the corresponding wells in the
Destination Plate, being careful to not create bubbles.

5.

Incubate the cells for 4-24 hours (as determined during transduction optimization).

6.

Add 75 μL of preferred culture medium to cells without removing medium. Make the necessary adjustments toserum concentration so that the final serum concentration matches the Preferred Culture Medium after adding
to wells.

7.

After 24 hours, replace medium with Preferred Culture Medium supplemented with 1 μg/mL of freshly dissolved
doxycycline. C

8.

Culture cells for an additional 48-72 hours (as determined during transduction optimization).

9.

Choose one well in the Destination Plate for counting fluorescence-expressing colonies of cells. This should bea well in which individual colonies of cells can be visualized and counted. Count each multi-cell colony as one
transduction event, as the cells have been dividing over the culture period (Figure 10). Calculate the average numberof fluorescent-positive colonies from the same destination well of each replicate.

Doxycycline is unstable in solution. It is recommended that a fresh working solution be stored for no more than three days at 4 °C.
Additionally, when inducing cells with doxycycline, the medium should be replaced with fresh medium containing fresh doxycycline
every 24-48 hours.
18

shMIMIC Inducible Lentiviral microRNA
Technical Manual
Make five-fold dilutions into
Dilution Plate according toprotocol and Table 2.

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

A
B
C
D
E
F
G
H

Remove medium fromdestination plate.
Add 25 µL diluted lentiviralparticles from each well in the
Dilution Plate to cells in thecorresponding well of the
Destination Plate

A
B
C
D
E
F
G
H

Dilution plate

Figure 9. Diagram for dilution series oflentiviral particles (Dilution Plate) andaddition to cells (Destination Plate).

Destination plate

Table 3. Example setup for lentiviral particle dilution series.

Dilution Plate
Well

Lentiviral particle serial
dilution volume

Volume ofdilution medium

A1
A2
A3
A4
A5
A6
A7
A8

10 µL (*control)
20 µL (from A1)
20 µL (from A2)
20 µL (from A3)
20 µL (from A4)
20 µL (from A5)
20 µL (from A6)
20 µL (from A7)

40 µL
80 µL
80 µL
80 µL
80 µL
80 µL
80 µL
80 µL

Dilution factor

Volume of diluted lentiviralparticles used in transduction(Destination Plate)

5
25
125
625
3125
15625
78125
390625

25 µL
25 µL
25 µL
25 µL
25 µL
25 µL
25 µL
25 µL

*Control (non-silencing shRNA control lentiviral particles). Repeat identical dilution series in wells B1 to B8.

Functional titer in transducing units per mL (TU/mL) can be determined using thefollowing formula:
Number of TurboGFP-positive colonies × Dilution factor (Table 2) ÷ 0.025 mL (Volume of diluted
lentiviral particles used) = Functional titer of non-silencing control lentiviral particles stock inyour cell line (TU/mL)
Relative transduction efficiency of your cell type can be determined by using the following formula:
Functional titer of non-silencing control in your cell line (TU/mL) ÷ Titer of non-silencing control
lentiviral particles stock as calculated by Dharmacon in HEK293T (TU/mL) (reported on the
C of A) = Relative transduction efficiency of your cell line
Figure 10. Example of individual colonies
in HEK293T cells 72 hour post-transduction.
Four colonies are circled. Imaged at 40x
magnification.

Use the calculated relative transduction efficiency of your cell line to calculate the anticipatedfunctional titer for each lentivirus using the following formula:
Relative transduction efficiency of your cell line × Titer of the lentivirus as calculated by in
HEK293T cells (TU/mL) = Anticipated functional titer in your cell line (TU/mL)

Calculating examples
If you counted 58 fluorescent-positive colonies in well A7 of the destination plate, the titer of the non-silencing control
lentiviral particles in your cell line would be calculated as follows:
58 (transduction positive colonies) × 78,125 (dilution factor) ÷ 0.025 mL (volume of diluted lentiviral particles
used) = 1.8 × 108 TU/mL functional titer of non-silencing control in your cell line
If the titer for the non-silencing control lentiviral particles on the product insert was listed as 9.0 × 108 TU/mL, the relativetransduction efficiency of your cell type would be determined as follows:
1.8 × 108 TU/mL (functional titer in your cell line) ÷ 9.0 × 108 TU/mL (titer as indicated on product insert)= 0.2 relative transduction efficiency
If the relative transduction efficiency of your cell line is 0.2 and the titer of a lentiviral stock, as indicated on the C of A, is
5.0 × 108 TU/mL, the anticipated functional titer of the pool in your cell line would be determined as follows: 0.2 (relative transduction
efficiency) × 5.0 × 108 TU/mL (titer as indicated on product insert) = 1.0 × 108 TU/mL anticipated functional titer in your cell line
19

shMIMIC Inducible Lentiviral microRNA
Technical Manual

TurboGFP and TurboRFP expression from a single lentiviral integration can be difficult to see without a high-quality fluorescencemicroscope. Additionally, phenol red absorbs at both the excitation and emission wavelengths of both of fluorophores. To enhancethe visibility of fluorescing cells, replace the culture medium with a medium that is low in phenol red.

Calculating the volume of lentiviral particles to use in a transduction
The functional titer of each lot of shMIMIC Inducible microRNA Lentiviral Particles is given in the Certificate of Analysis(C of A). However, lentiviral transduction efficiency varies widely and depends on factors such as target cell type, presence/
absence of serum during transduction, duration of exposure to lentiviral particles, presence/absence of transduction
enhancers, composition of transduction medium and others. Therefore, for sensitive applications where an MOI of 0.1-0.3
is desired, it is recommended that for each batch of unique cell type and for each variation in transduction conditions, thecorrect volume of lentiviral particles should be empirically determined using control particles.

Optimizing doxycycline concentration
The Tet-On 3G induction system employed in the SMARTchoice Inducible Lentiviral system generally allows robust
shMIMIC microRNA induction at doxycycline doses between 0.1 µg/mL and 1.0 µg/mL, although this depends on thecell type in use. Furthermore, intermediate expression of the shMIMIC microRNA, may be achievable at doses between
10 ng/mL and 100 ng/mL, although this will require optimization to obtain consistent results. In some cell types, doses of
doxycycline above 0.5 µg/mL have been observed to affect cell viability. We strongly recommend that for each cell type,a doxycycline dose curve should be performed to identify the lowest effective concentration of doxycycline resulting in
the least effect on cell viability.
SMARTvector SMARTvector
inducible
inducible
non-targetGAPDH or
ing control PPIB control

Volume oftransduction
medium:1

Figure 11. Plate layout for transductions using
SMARTvector Inducible Positive and Negative
Control shRNAs.

90 μL

A

80 μL

B

80 μL

C

80 μL

D

80 μL

E

80 μL

F

80 μL

G

80 μL

H

2

3

4

5

6

7

8

9

10 11 12No lentiviralparticles in Row H

The following protocol uses the SMARTvector Inducible Negative and Positive RNAi Controls to optimize the doxycycline
dose for both maximal expression and maximal cell viability. This experiment is recommended whenever shMIMIC Inducible
microRNAs are used in a new cell line or type.

20

shMIMIC Inducible Lentiviral microRNA
Technical Manual

Identifying the optimal doxycycline dose for the cells of interest
Day 1:
Plate cells in biological triplicates in a 96-well culture plate at optimal cell density in appropriate growth medium as
determined in previous experiments (Chapter 3). If working with suspension cell lines, plate cells the day of transduction(see protocol below for recommendations).
Day 2:
1.

Thaw the SMARTvector Inducible positive and negative control shRNA lentiviral particles on ice.

2.

For both the positive and negative controls, prepare enough transduction medium with lentiviral particles totransduce 24 wells of a 96-well plate at an MOI of 0.3.

3.

Remove culture plates from incubator and replace medium with 25 μL of transduction medium with lentiviralparticles according to the plate layout in Figure 11.

4.

Return culture plate to incubator.

5.

After 6-20 hours (as determined in Chapter 3), add 100 μL of preferred culture medium directly to each well without
removing medium. Make adjustments to serum concentration so that final serum concentration matches Preferred
Culture Medium after adding to wells.

Day 3:
Replace medium on cells with fresh preferred culture medium.
Days 4, 6 and 7:
Replace medium on cells with fresh preferred culture medium supplemented with the optimal dose of puromycin(determined in Chapter 3) for the target cell line.
Day 8:
1.

Ensure that puromycin selection is complete by examining Row H and determining that no viable cells remain.

2.

Replace culture medium on cells with fresh preferred culture medium supplemented with 0 ng/mL, 10 ng/mL,
50 ng/mL, 100 ng/mL, 250 ng/mL, 500 ng/mL, 1 µg/mL of freshly dissolved doxycycline (Figure 12).

Day 10:Replace culture medium on cells with fresh preferred culture medium, maintaining doxycycline at the doses
from Day 8.
SMARTvector SMARTvector
inducible
inducible
non-targetGAPDH or
ing control PPIB control

Doxycycline
dose:

1

Figure 12. Plate layout for optimizing induction of
expression at multiple doxycycline concentrations
following transductions using SMARTchoice Inducible
Positive and Negative Control shRNAs.

No DoxA

10 ng /mLB

50 ng /mLC

100 ng /mLD

250 ng /mLE

500 ng /mLF

1 μg /mL

G

2

3

4

5

6

7

8

9

10 11 12H
No lentiviralparticles in Row H
21

shMIMIC Inducible Lentiviral microRNA
Technical ManualDay 11:1.

Test for toxicity using alamarBlue™ or a similar assay for viability.

2.

Determine knockdown of GAPDH or PPIB positive control genes at the mRNA level with RT-qPCR relative tonegative control.

3.

Choose the doxycycline dose that results in maximal GAPDH or PPIB gene silencing while having a minimal
effect on cell viability. This doxycycline dose will be appropriate for all shMIMIC inducible microRNAs of the same
vector configuration in the tested cell type.

Establishing stable cell populations
For transformed cells, immortalized cells, or cell lines with unlimited proliferative capacity, stable cell lines may beestablished by transduction of shMIMIC inducible microRNAs lentiviral particles and subsequent puromycin selection.
These cell lines may be maintained in an uninduced state until expression of the shMIMIC microRNAs is desired. Similarly,
some primary cell types capable of 10 or more population doublings may also be used to establish inducible cell systems,
but these cells must be used within a limited number of passages from completion of selection, depending on thecharacteristics of the cell type.

Establishing a stable cell line
Day 1:
Transduce cells at a low MOI of 0.1-0.3 in a 96-well, 24-well or larger format.
Day 2:
Change growth medium.
Day 3:
Trypsinize cells and expand into a larger culture vessel with medium containing the predetermined concentrationof puromycin sufficient to select for stable integrants.
Day 5:
Change growth medium, maintaining the puromycin concentration used on Day 3.
Day 7 or later:
After cells have been selected for two to four days in puromycin, it is no longer necessary to maintain selection. Expand theculture until an adequate cell number has been achieved to freeze and archive the cell line. At this point the cell line is readyfor subsequent experiments.
Induce shMIMIC microRNA expression in the stable cell line using the optimal doxycycline dose (in the range of 0.1 µg/mL
and 1.0 µg/mL). TurboGFP or TurboRFP expression will become visible in about 24 hours, and will reach a maximum at
48 to 72 hours. Effects of mature microRNA expression on target gene repression at the mRNA and/or protein level can beassessed 72 hours after induction. Phenotypic assay timepoints will need to be optimized depending on assay.

Inducible shMIMC microRNA expression without establishing stable cell lines
It is not necessary to establish stable cell lines with shMIMIC Inducible microRNAs to perform a a mature microRNAgain-of-function experiment. Depending on the phenotype to be assayed, immortalized, primary and non-dividing cells
may be transduced with shMIMIC Inducible microRNAs Lentiviral Particles selected for two to four days in puromycin,
induced with doxycycline and assayed within a 7 to 10 day timeframe. Using inducible mature microRNA expression
systems in this context allows temporal separation of phenotypes associated with lentiviral transduction and puromycin
selection from phenotypes resulting from mature microRNA over-expression.

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Plate cells at density optimized for the assay. Initial cell density will depend on the desired density at the time of phenotypic
analysis, growth rate of cells and desired experimental timeframe. If targeting an MOI between 0.1-0.3, be certain to factor incell loss resulting from death of untransduced cells during puromycin selection. Remember to plate additional wells with cells
that will be used as controls to monitor the puromycin selection.
Day 2:
Transduce cells at an MOI of 0.1-0.3 in 96-well or 24-well format.
Day 3:
Replace culture medium.
Day 4:
Replace culture medium with fresh medium containing a puromycin concentration that has been determined to kill
untransduced cells in two to three days.
Day 6 or 7:

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1.

Examine control cells (untransduced) and determine if puromycin selection is complete. If live cells remain in theuntransduced wells, continue puromycin selection for another day.

2.

Depending on the phenotype to be examined, doxycycline induction may be started at this time. We have
not observed any negative effects of continuing puromycin selection while beginning doxycycline induction.
Alternatively, the medium may be replaced with fresh growth medium and doxycycline induction may be initiated at
a later time point.

3.

Induce shMIMIC microRNA expression in the cells using the optimal doxycycline dose. TurboGFP or TurboRFP
expression will become visible at 24 hours and will reach a maximum at 48 to 72 hours. Similarly, microRNA target
gene repression at the mRNA and/or protein level is typically observable 72 hours post-induction. Longer periodsof exposure to doxycycline may be necessary to achieve some phenotypes.

AppendixLentiviral packaging
During the preparation of lentiviral particles, the transfer plasmid (containing the specific shMIMIC microRNA of interest)
and the helper plasmids (containing the elements essential for viral packaging) are co-transfected into the viral packaging
cell line, such as HEK293T. The lentiviral vector genomes are produced, encapsulated and released as virion particles fromthe cells into the surrounding medium. Transduction of cells with this lentiviral supernatant (medium containing thelentiviral particles) can be performed at low or high MOIs. Lower MOIs may be used for more easily transduced cells or
when establishing stable cell lines. High MOIs may be needed for difficult-to-transduce cell lines or in vivo applications;however, it is important to note that addition of large volumes of low-titer lentiviral supernatant can often be toxic. This isdue in part because the lentiviral supernatant also contains cellular debris (metabolites, nucleases and proteases) derived
from the packaging and production process that will affect the viability of transduced cells. Recognizing the limitations
and requirements for lentiviral preparations, we have developed a proprietary production and manufacturing process
leveraging the Dharmacon Trans-Lentiviral Packaging System that effectively concentrates lentiviral particles while at thesame time greatly reduces packaging cell debris. Researchers receive preparations of purified, high-titer lentiviral particles(≥ 1 × 107 TU/mL).

Lentiviral particle titering
Prior to shipping, SMARTvector Inducible lentiviral particles are titered in one of two ways. The particles are either
directly functionally titered by transducing HEK293T cells with serial dilutions of lentiviral particles, inducing TurboGFP
expression with doxycycline, and then quantifying TurboGFP-positive cells by flow cytometry. Alternatively, a p24 ELISA isused to measure lentiviral particle titer. Because p24 titers overestimate functional titers, a vector-matched SMARTchoice23

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Inducible reference control of known functional titer is included in the p24 ELISA. The functional titer ofthe reference control is determined by flow cytometry as previously described. Including the reference control in the
ELISA allows the p24 titer to be converted to a functional titer by correlating the p24 values of the sample with thereference control.

Stability and storageshMIMIC Inducible microRNA lentiviral particles are shipped on dry ice as 25 μL aliquots and must be stored at -80 °C.
Under these conditions, lentiviral particles are stable for at least 12 months. Repeated freeze-thaw cycles should be
avoided, as this is expected to negatively affect titer. Once thawed, unused lentiviral particles should be kept on ice,
aliquoted into smaller volumes (if necessary) and immediately returned to -80 °C.

Quality assurance and control
shMIMIC Inducible microRNA Lentiviral Particles are subject to stringent quality control at multiple steps during the
manufacturing process, including:
1.

Sanger sequencing of each clone to ensure integrity of shMIMIC microRNA sequence;2.

lentiviral particle titering using flow cytometry or p24 ELISA, followed by conversion to a functional titer based ona matched-vector reference control of known functional titer;3.

confirmation of fluorescent reporter expression (TurboGFP or TurboRFP) following transduction and doxycycline
induction in HEK293T cells;

4.

examination of each batch to ensure preparations are free from mold and bacterial contamination; and5.

generation of Certificate of Analysis (C of A) with specified lentiviral titers for each batch included witheach shipment.

Trans-lentiviral packaging system biosafety features
Historically, the greatest safety risk associated with a lentiviral delivery platform stems from the potential generationof recombinant lentiviruses that are capable of autonomous replication. Dharmacon minimizes these hazards to thegreatest degree by combining a disabled lentiviral genome with the proprietary Trans-Lentiviral Packaging System.
Starting with the pNL4-3 molecular clone of HIV1 (GenBank Accession Number AF324493), the lentiviral backbone has
been modified to eliminate all but the most essential genetic elements necessary for packaging and integration (5' LTR,Psi sequences, polypurine tracts, Rev responsive elements and 3' LTR). Furthermore, all essential replication genes (gag,pol and env) and accessory genes (tat, rev, vif, vpr, vpu and nef) have been removed. Therefore, all lentiviral particles
produced by Dharmacon are replication-incompetent, containing less than 30% of the wild-type HIV-1 genome.
Additionally, the 3' LTR has been modified to remove the enhancer elements located in the U3 region. These enhancer
elements are needed for native transcriptional activity from the lentiviral LTR. The deletion within the 3'-LTR (ΔU3) results
in a vector that is self-inactivating (SIN), which greatly reduces the probability of producing recombinant particles andlimits cellular toxicity often associated with expression from the lentiviral LTR. Additional safety features are incorporated
by the manufacturing process itself. Generation of lentiviral particles requires a packaging step during which theexpression construct containing the shRNA sequence is encapsulated into a viral particle structure. Lentiviral vector
packaging systems have divided the essential functions amongst multiple plasmids to reduce the risk of generating
replication-competent lentivirus (RCL). The split-genome packaging system is designed so that multiple recombination
events between the components are required to generate a lentiviral particle capable of autonomous replication.
Commercially available 3rd generation lentiviral vector systems separate the envelope, env (such as VSVg) from the gagpro-pol, which encodes structural and enzymatic functions. The Trans-Lentiviral Packaging System provides an evenhigher level of safety over 3rd generation packaging systems by further splitting the lentiviral pol [reverse transcriptase(RT) and integrase (IN) functions] from gag-pro. Because the RT and IN enzymes are provided in trans to Gag-Pro,
additional recombination events are necessary to produce RCLs. Among commercially available lentiviral vector systems,
the Trans-Lentiviral Packaging System offers a superior safety profile as the packaging components are separated onto
five plasmids. Additionally, expression of gag-pro and tat-rev are under the control of the conditional tetracyclineresponsive promoter element (TRE), limiting expression of these lentiviral components strictly to the packaging cell line.
A detailed description of the Trans-Lentiviral Packaging System can be found in (Wu, et al. 2000).
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Frequently asked questions
Do I have to select my cells with puromycin?
This depends on the how shMIMIC Inducible microRNAs are used. We recommend transducing your cells at a target MOI
between 0.1 and 0.3. At low MOIs such as these, the majority of cells will not have a proviral genome integrated into theirgenomic DNA immediately following transduction. Therefore puromycin selection is necessary to remove untransduced
cells from the population, resulting in a cell population in which the majority of the remaining cells carry a single viral
integration. Puromycin selection may not be necessary if cells are transduced
at high MOI.
What happens if I transduce at higher MOI than recommended (> 1)?
For some limited applications, a higher rate of lentiviral integration may be preferred. Each copy of the TRE3G promoter
will contribute some low-level of basal shMIMIC microRNA expression. Therefore at higher MOI, more “leaky” shMIMIC
microRNA expression may be observed. In addition, Tet-On proteins, including Tet-On 3G have been shown to be toxic
when highly expressed. Higher MOI transductions will result in higher Tet-On 3G expression, and potentially more striking
effects on cell viability. Monitoring for these effects is suggested if higher MOIs are used.
How do I choose the best promoter for my cells?
We have found the mCMV- and PGK-based vectors to be functional in most cell types. However, in cells such as mouseembryonic stem cells, we have found the mEF1α and hEF1α vectors to be more effective. To ensure functionality inyour cell type of interest, please use the SMARTchoice Inducible Non-targeting Control 4-Pack to assess the cell-specificactivity of the four promoter options.
Can I culture my cells in doxycycline continuously to treat this as a constitutive knockdown vector?
Yes.
How long can I passage my cells in culture before inducing expression with doxycycline?
We have observed shMIMIC Inducible microRNA over-expression in stable cell populations to be consistent over 20
passages, even with just a single initial puromycin selection period. However, long-term silencing of the lentiviral content
may occur in some cell types. Therefore it is generally good practice to freeze early passages of the cell population, anduse the cells within a limited passage number.
Do I need to generate a stable cell line expressing Tet-On first?
No, shMIMIC Inducible microRNA Lentiviral Particles carry all necessary elements for selection and inducible expression.
Cell lines capable of inducible expression are generated with a single transduction.
Since there is little or no shMIMIC microRNA expression in the absence of doxycycline, why do I need to use
a Non-targeting Control shRNA?
Although doxycycline is generally accepted as “inert,” some cellular phenotypes, such as reduced growth rate, may
be observed upon exposure to doxycycline. In addition, fluorescent reporters such as TurboGFP and TurboRFP can causesome subtle changes in cellular phenotypes. To rigorously control for both of these effects, it is necessary to
use a vector-matched Non-Targeting Control shRNA to identify true mature microRNA-dependent phenotypes.
Can I use tetracycline to induce expression of the mature microRNA in shMIMIC Inducible microRNAs?
No, Tet-On 3G has been optimized for binding doxycycline, and it responds poorly to tetracycline.

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8

References
1.

Bartel DP, MicroRNAs: target recognition and regulatory functions. Cell. 136(2), 215-33 (2009).

2.

Curtin JA, Dane AP, et al., Bidirectional promoter interference between two widely used internal heterologous
promoters in a late-generation lentiviral construct. Gene Ther. 15(5), 384-90 2008).

3.

Kappes JC, Wu X, Safety considerations in vector development. Somat. Cell Mol. Genet. 26(1-6), 147-58 (2001).

4.

Kim VN, MicroRNA biogenesis: coordinated cropping and dicing. Nat. Rev. Mol. Cell Biol. 6(5),376-85 (2005).

5.

Kim VN, Han J, Biogenesis of small RNAs in animals. Nat. Rev. Mol. Cell Biol. 10(2), 126-39 (2009).

6.

Krutzfeldt J, Rajewsky N, Silencing of microRNAs in vivo with 'antagomirs'. Nature. 438, 685-689 (2005).

7.

Lee R, Feinbaum R, The C. elegans heterochronic gene lin-4 encodes small RNAs with complementarity
to lin-14. Cell. 75(5), 843-54. (1993).

8.

Loew R, Heinz N, et al., Improved Tet-responsive promoters with minimized background expression.
BMC Biotechnol. 10, 81 (2010).

9.

Miyoshi H, Blomer U, Development of a Self-Inactivating Lentivirus Vector. J. Virol. 72(10), 8150-8157 (1998).

10. Morimoto M, Kopan R. rtTA toxicity limits the usefulness of the SP-C-rtTA transgenic mouse.
Dev. Biol. 325(1), 171-178 (2009).
11. Sisson TH, Hansen JM, et al., Expression of the reverse tetracycline-transactivator gene causes emphysema-likechanges in mice. Am. J. Respir. Cell Mol. Biol. 34, 552-560 (2006).
12. Whitsett JA, Perl AK. Conditional control of gene expression in the respiratory epithelium: A cautionary note.
Am. J. Respir. Cell Mol. Biol. 34, 519-520 (2006).
13. Wightman B, Ha I, Posttranscriptional regulation of the heterochronic gene lin-14 by lin-4mediates temporal patternformation in C. elegans. Cell. 75(5), 855-62 (1993).
14. Wu X, Wakefield JK, Development of a novel trans-lentiviral vector that affords predictable safety.
Mol. Ther. 2(1), 47-55 (2000).

9Limited use licenses
The Products, use and applications, are covered by pending and issued patents. Certain Label licenses govern the useof the products, these can be found at Dharmacon Licensing Statements. It is each Buyer’s responsibility to determine
which intellectual property rights held by third parties may restrict the use of Products for a particular application. Please
review the Label Licenses governing all use of the Products.

If you have any questions, contact

alamarBlue, Hyclone, and Nunc are registered trademarks of Thermo Fisher Scientific, Inc. CellTiter-Blue is a trademark of Promega Corporation. Lysol is a
registered trademark of Reckitt Benckiser Group. Tet-On is a trademark of Clontech Laboratories, Inc. TurboGFP and TurboRFP are trademarks of Evrogen Inc.
All trademarks are the property of Horizon Discovery Company unless otherwise specified. ©2018 Horizon Discovery Group Company—All rights reserved.
First published April 2014. UK Registered Head Office: Building 8100, Cambridge Research Park, Cambridge, CB25 9TL, United Kingdom.

V51018t +44 (0) 1223 976 000 (UK) or +1 800 235 9880 (USA); +1 303 604 9499 (USA)f + 44 (0)1223 655 581
w horizondiscovery.com/contact-us or dharmacon.horizondiscovery.com/service-and-support
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom

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