tGFP

IRES

PuroR

RRE

WPRE

shRNA

3' SIN LTR

5' LTRpSMART 2.0

TECHNICAL MANUAL

AmpR

pUC ori

SV40 ori

Precision LentiORF™ Collection
U6

hPGK

shRNA

PuroR

RRE
3' SIN LTR

RSV/5' LTR

pLKO.1

RSV/5ʹ LTR

Ψ

Ψ

RRE
Cat #OHS5832, 5833, 5834,
5898, 5900

5ʹ LTRΨΨ

RRE

Important safety note

Product description

Please follow the safety guidelines for use and production of vector-based
lentivirus as set by your institution’s biosafety committee.
pUC ori
AmpR• For glycerol stocks of E. coli containing lentiviral plasmids, BSL1
guidelines should be followed• For handling and use of lentiviral products to produce lentiviralparticles, BSL2 or BSL2+ guidelines should be followed• For handling and use of lentiviral particle products, BSL2 or
BSL2+
hCMV
guidelines should be followed

The Precision LentiORF collection is a library of expression-ready lentiviral open
reading frames (ORFs) generated using gene content from the fully sequenced
ORFeome Collaboration Collection. Its unique design expresses the ORF, thefluorescent reporter and the selection marker from one promoter, providing avisual indicator for ORF expression and a mechanism for selecting transduced
cells (Figure 1 and Table 1). When used in lentiviral format, precise control
of expression level can be achieved by varying the multiplicity of infection(MOI). This collection provides a robust ORF expression tool for gene analysis.
Highlights include:R

IRES

microRNA

Puro

WPRE

• Versatile vector design with convenient cloning site for the additionof fusion tags (Figure 2)• Transfection or transduction options for ORF expression• Dual marker cassette to optimize transduction and maximize expression ofyour ORF in the cell population- Nuclear localized TurboGFP™ (Evrogen™, Moscow, Russia) for easy tracking- Blasticidin S resistance for selection• Lentiviral transduction extends gene expression to a broad rangeori
of SV40cell types• Fully sequenced ORFs for increased confidence in gene expression results

3' SIN LTRΨpSMART 2.0

5' LTR**Our Precision LentiORFs are available in glycerol stock or viral particle
format. If viral particle format is purchased, begin work with Protocol IX –Determining Relative Transduction Efficiency on page 11.

AmpR

RRE

Ψ

tGFP

pUC ori5ʹ LTR

RRE

Please note that Precision LentiORF vectors are not compatible with third
generation packaging systems, due to the requirement of the expression oftat, which third generation systems do not contain. We recommend theDharmacon™ Trans-Lentiviral™ Packaging System for use with our vectors.

hCMV
tGFPnuc-2a-

Blast R

RRE

WPRE

Ψ

IRES

Ψ

Multi Tag
Cloning Site

3' SIN LTR

5' LTR

pLOC
AmpR

pUC ori

SV40 ori

Figure 1. pLOC lentiviral ORF clone.

SMARTchoicepromoters

hCMV

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mCMV
hEF1α
mEF1α
CAG

SMARTchoice shRNA

5ʹ LTRORF

Table 1. Features of the pLOC vector.

Antibiotic resistancepLOC contains two resistance markers (Table 2).

Vectorelement

UtilityhCMV

Human cytomegalovirus promoter drives strongtransgene expression

ORF

Full-length human ORFs from the ORFeome
Collaboration Collection

Multi Tag
Cloning site

Convenient cloning site for the addition of a purification
or tracking tag

IRESInternal ribosomal entry site allows expression of
Evrogen™ TurboGFP™ and Blasticidin S resistance genes
in a single transcript

tGFPnuc

TurboGFP reporter expressed in the cell nucleus to facilitate
visual tracking of transduction and expression

Blast R

Blasticidin S resistance permits antibiotic-selective pressure
and propagation of stable integrants

2a

2a self-cleaving peptide allows simultaneous expressionof tGFPnuc and Blasticidin S resistance proteins from asingle transcript

5' LTR

5' long terminal repeat

3' SIN LTR

3' self-inactivating long terminal repeat for increased lentivirus
safetyΨ

Psi packaging sequence allows viral genome packaging using
lentiviral packaging systems

WPRE

Woodchuck hepatitis posttranscriptional regulatory element
enhances transgene expression in the target cells

pLOC
TurboRFP
9.9kb

Antibiotic

Concentration

Utility

Fisher Scientific Cat #Ampicillin(carbenicillin)

100 μg/mL

Bacterial
BP2648-250
selection marker(outside LTRs)

Blasticidin S

variable

Mammalian
selectable
markerBP2647-25

Quality control
Each ORF has been end-sequenced to verify identity following the cloning
process. The att site, MultiTag cloning site and secondary stop codons havealso been sequence verified.

Additional safety information
Historically, the greatest safety risk associated with a lentiviral delivery
platform stems from the potential generation of recombinant viruses thatare capable of autonomous replication. Our Precision LentiORF platform
minimizes these hazards to the greatest degree by combining a disabled viralgenome with the proprietary Dharmacon Trans-Lentiviral packaging process.
Starting with the HXB2 clone of HIV-1 (GenBank Accession Number K03455),
the lentiviral backbone has been modified to eliminate all but essential
genetic elements necessary for packaging and integration (including 5' LTR,Psi sequences, polypurine tracts, Rev responsive elements and 3' LTR). The
resultant self-inactivating (SIN) vector greatly reduces the probability of
producing recombinant particles and limits cellular toxicity often associatedwith expression of HIV genes.
Additional safety features are incorporated by the manufacturing processitself. Generation of Precision LentiORF lentiviral particles requires a
packaging step during which the expression construct containing the ORF
sequence is enclosed in a viral capsid. Gene functions that facilitate thisprocess (such as encoded by the structural genes gag, pol, env) are distributed
amongst multiple helper plasmids which do not contain significant regionsof homology. This tactic further minimizes the probability of recombination
events that might otherwise generate viruses capable of autonomous
replication. Among commercially available lentiviral vector systems, the TransLentiviral 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 viralcomponents strictly to the packaging cell line. A detailed description of theTrans-Lentiviral Packaging System can be found in Wu et al. (1).
With these safety measures in place, Precision LentiORF lentiviral particles
can be employed in standard Biosafety Level 2 tissue culture facilities. Any
investigator who purchases viral vector products is responsible for consultingwith their institution’s health and biosafety group for specific guidelines on
the handling of lentiviral vector particles. Further, each investigator is fully
responsible for obtaining the required permissions for the acceptance oflentiviral particles into their local geography and institution.

Figure 2. Detailed vector map of pLOC lentiviral vector containing the Evrogen TurboRFP
ORF. The lentiviral ORF clone contains attB1 and attB2; the ORF clone contains attL1
and attL2.

The specific sequence of the att site may vary between
clones.

In the U.S., download the U.S. Department of Health and Human Services
Centers for Disease Control and Prevention and National Institutes of Health,Biosafety in Microbiological and Biomedical Laboratories (BMBL), Fifth Edition,
Feb 2007 here: cdc.gov/biosafety/publications/index.htm
See also: NIH Guidelines For Research Involving Recombinant DNA Molecules(NIH Guidelines), downloadable here:
osp.od.nih.gov/biotechnology/nih-guidelines
For Biosafety Considerations for Research with Lentiviral Vectors, seeosp.od.nih.gov/wp-content/uploads/Lenti_Containment_Guidance

horizondiscovery.com

Protocol I – plasmid preparation

Table 3. Restriction digest reaction setup.

2 μL

DNA sample (up to 1 µg) in water

X μL

FastDigest EcoRI (Cat #FD0274)

1 μL

Final volume

20 μL

Protocol III – tagging Dharmacon precision
LentiORF with a C-terminal tag
Precision LentiORFs are available in “open” (the native stop codon has been
removed) and “closed” (the ORF retains its native stop codon) configurations(see clone information). Translation of the “closed” configuration ORFs
terminates at its natural stop codon immediately after last sense codon of the
ORF reference sequence.

Due to the tendency of viral vectors to recombine, we
recommend keeping the incubation times as short as
possible and avoid subculturing. Return to your original
glycerol stock for each plasmid preparation.

Protocol II – Precision LentiORF plasmid DNA

Analysis by restriction digest
The following is a sample protocol for restriction analysis of Precision
LentiORF plasmid DNA using Thermo Scientific™ FastDigest™ EcoRI
Restriction Enzyme (Cat #FD0274). EcoRI cuts 15 bases upstream of attB1 site
flanking the ORF at the 5' end (which corresponds to 30-50 nt upstream of
ORF start codon, depending on the clone configuration – see specific clone
information). A second EcoRI site is located 1348 bp downstream of attB2
site, approximately 1.37 kb downstream of the last sense codon of the ORF.
Alternatively, SpeI or PacI flanking attB1 site at the 5' end and NheI or AscI
flanking attB2 at 3' end in the vector may be used to cut out the fragment
corresponding to the ORF. Use of EcoRI is preferable for smaller ORFs.
1. Set up EcoRI restriction digest reaction as follows (Table 3).
2. Mix gently by pipetting.
3. Incubate at 37 °C for 5 minutes to digest.
4. Run cut and uncut plasmid on 1% agarose gel (Figure 3).

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LadderCommon vector band

Culture conditions for 96-well bio-block plasmid preparation
1. Prepare a 96-well bio-block with 1 mL per well of 2x LB broth (low salt)
medium containing 100 μg/mL carbenicillin.
2. Inoculate each well with 1 µL of the glycerol stock culture.
3. Incubate at 37 °C with shaking (~ 170–200 rpm) for 18–19 hours to
produce good plasmid yield. For plasmid preparation, follow the
protocol recommended by the plasmid isolation kit manufacturer.
96-well bio-block plasmid preparation protocol in
conjunction with an appropriate 96-well plasmid purification
method are used to purify plasmid DNA. Combine thecultures from two bio-blocks prior to purification, and elutewith water at the final step.

ORF2
Cut

ORF1

Uncut

Most plasmid miniprep kits recommend a culture volume of 1–10 mL
for good yield.
1. Upon receiving your glycerol stock(s) containing the ORF of interest
store at –80 °C until ready to begin.
2. To prepare plasmid DNA, first thaw your glycerol stock culture andpulse vortex to resuspend any E. coli that may have settled to thebottom of the tube.
3. Take a 10 μL inoculum from the glycerol stock into 3–5 mL of 2x LB broth(low salt) medium with 100 μg/mL carbenicillin. Return the glycerol
stock(s) to –80 °C.
4. Note: If a larger culture volume is desired, incubate the 3-5 mL culture
for 8 hours at 37 ˚C with shaking and use as a starter inoculum.
Dilute the starter culture 1:500-1:1000 into the larger volume.
5. Incubate at 37 °C for 18-19 hours with vigorous shaking.
6. Pellet the 3–5 mL culture and begin preparation of plasmid DNA.

10x Thermo Scientific™ FastDigest™ buffer

Cut

20 g/L
10 g/L
5 g/L
100 μg/mL

AmountX μL

Uncut

2x LB broth (low salt) medium preparation:
LB-Broth-Lennox (Fisher Scientific Cat #BP1427500)
Peptone(Fisher Scientific Cat #BP9725–2)
Yeast Extract (Fisher Scientific Cat #BP1422–500)
Carbenicillin (Fisher Scientific Cat #BP2648–250)

Component
Water, nuclease-free (Cat #R0581)

LadderCulture conditions for individual plasmid preparations
For plasmid preparation, grow all Precision LentiORF clones at 37 °C in
2x LB broth (low salt) medium plus 100 μg/mL carbenicillin.

ORF specificbands

Figure 3. Restriction analysis of two Precision LentiORF clones with differen sized ORFs.
Lanes 1 & 6 are a 10 kb molecular weight ladder (10 kb, 7 kb, 5 kb, 4 kb, 3 kb, 2.5 kb, 2
kb, 1.5 kb, 1 kb). Lanes 2 and 4 show uncut plasmid. Lanes 3 and 5 are EcoRI digests. The
white arrow points to the 7.8 kb band common to the back bone of all Precision LentiORF
clones. The lower bands (ORF1 producing a 2.8 kb band and ORF2 producing two bands,
2.5 kb and 1.3 kb) are specific to each ORF. ORF2 has an additional EcoRI site within it
producing two ORF specific bands noted with the asterisks.

“Open” configuration of the ORFs was created to allow for C-terminal fusionsof the ORFs with the tag of interest. In these ORFs, translation terminates atthe stop codon in the vector immediately downstream of the attB2 site adding
11–16 amino acids to its C-terminus (Figure 4). If desired, the stop codons in
the vector can be replaced by the fusion tag at Nhe/AscI sites flanking the stopcodon in the vector. Care should be taken to place the fusion tag in the same
reading frame as the stop codon.

ORF
attB2.1 site
Stop condons
ATG GAC GAG CTG TAC AAG TAC CCA ACT TTC TTG TAC AAA GTG GTT GCT AGC TAA TGA
Frame of translation

NheI

*

*

ACCGGGCGCGCC
GGCGCGCC CCGCCCCTCTC
AscI

Figure 4. Example of 3' end of ORF and flanking sequence. Highlighted gray are NheI
and AscI restriction sites flanking ORF-attB2 site at the 3' end. Highlighted blue is theattB2 site (version attB2.1) Underlined is an invariable part of the attB2 site and theadjacent vector sequence. The secondary stop codons have asterisks beneath them.

Protocol IV – Blasticidin S selection
Blasticidin S kill curve and selection
The pLOC vector confers resistance to Blasticidin S in transduced or transfected
cells. Blasticidin S selection can be used to eliminate non-transduced or
non-transfected cells. In order to generate stable cell lines, it is important
to determine the minimum amount of Blasticidin S required to kill nontransfected or non-transduced cells.
We recommend testing your cell line of choice by generating a Blasticidin S killcurve following the protocol below. The general concentration range for manycells is 5–15 µg/mL.
Blasticidin S kill curve
1. On day 0, plate 5 × 104 cells per well in a 24-well plate in enough wells
to carry out your Blasticidin S dilutions. Incubate overnight.
2. Prepare medium specifically for your cells containing a range ofantibiotic, for example: 0–20 μg/mL Blasticidin S.
3. The next day (day 1) replace the growth medium with the medium
containing dilutions of the antibiotic into the appropriate wells.
4. Incubate at 37 °C, 5% CO2.
5. Approximately every 2-3 days replace with freshly preparedselective medium.
6. Monitor the cells daily and observe the percentage of surviving
cells. Optimum effectiveness should be reached in 3–6 days underBlasticidin S selection.
7. The minimum antibiotic concentration to use is the lowest
concentration that kills 100% of the cells in 3–6 days from the startof antibiotic selection.

1. The day before transduction, seed a 24-well tissue culture plate with
HEK293T cells at 5 × 104 cells per well in DMEM (10% FBS, 1% pen-strep).
The following day, the well should be no more than
40-50% confluent.
2. Make dilutions of the viral stock in a round bottom 96-well plate using
serum-free medium. Utilize the plate as shown in Figure 5 using one row
for each virus stock to be tested. Use the procedure below (starting atstep 4) for dilution of the viral stocks. The goal is to produce a series offive-fold dilutions to reach a final dilution of 390,625-fold.
3. To each well add 80 μL of serum-free medium.
4. Add 20 μL of thawed virus stock to each corresponding well incolumn 1 (five-fold dilution). Pipette contents of well up and down
10-15 times. Discard pipette tip.
5. With new pipette tips, transfer 20 μL from each well of column 1 to thecorresponding well in column 2. Pipette contents of well up and down
10–15 times. Discard pipette tip.
6. Repeat transfers of 20 μL from columns 2 through 8, pipetting up
and down 10–15 times and changing pipette tips between eachdilution. Pre-incubate the dilutions of the virus stock for 5 minutes atroom temperature.
7. Label the 24-well plate as shown in Figure 6 using one row for eachvirus stock to be tested.
It is strongly recommended that you use a high quality
multichannel pipettor when performing multiple dilutions.

Five-fold Dilutions

Protocol V – transfection
For transfection of Precision LentiORF plasmids we recommend using
DharmaFECT kb. Conditions should be optimized for your cell line of interest.
For more details see our protocol.

Protocol VI – packaging lentivirus
The pLOC vector is tat dependent, so you must use a packaging system that
expresses the tat gene. For packaging lentiviral constructs, we recommend
the Trans-Lentiviral Packaging System. The Trans-Lentiviral Packaging Systemallows creation of a replication-incompetent, HIV-1-based lentivirus which
can be used to deliver and express your gene of interest in either dividing or
non-dividing mammalian cells. The Trans-Lentiviral Packaging System uses
a replication-incompetent lentivirus based on the translentiviral systemdeveloped by Kappes et al2. For protocols and information on packaging
Precision LentiORFs with our Trans-Lentiviral Packaging System, please see theproduct manual available on our website. Here

Protocol VII – viral titering
Viral titering
Follow the procedure below to determine the titer of your lentiviral stock using
the mammalian cell line of choice if you have produced viral particles yourself.
This protocol uses the Dharmacon HEK293T cell line that is available as part of
our Trans-Lentiviral ORF Packaging Kit (Cat #TLP5918). You can use a standard
Dharmacon HEK293T cell line as
an alternative.
If you have generated a lentiviral stock of the expression
control (such as Precision LentiORF RFP control ), we
recommend titering this stock as well.

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1
Virus stock 1
Virus stock 2
Virus stock 3
Virus stock 4

2

3

4

5

6

7

8

9 10 11 12

A
B
C
D
E
F
G
H

Figure 5. Five-fold serial dilutions of virus stock .

1

2

3

4

5

6

Replicate 1 A
Replicate 2 B
Replicate 3 C
Replicate 4 D
Figure 6. A twenty-four well tissue culture plate seeded with Dharmacon HEK293T cells isused to titer the virus.

8. Remove culture medium from the cells in the 24-well plate.
9. Add 225 μL of serum-free medium to each well.
10. Transduce cells by adding 25 μL of diluted virus from the original
96-well plate (Figure 5) to a well on the 24-well destination plate(Figure 6) containing the cells. For example, transfer 25 μL from wellA2 of the 96-well plate into well A1 in the 24-well plate (Table 4).
11. Incubate transduced cultures at 37 °C for 4 hours.
12. Remove transduction mix from cultures and add 1 mL of DMEM
(10% FBS, 1% pen-strep).
13. Culture cells for 48 hours.
14. Count the TurboGFP expressing cells or colonies of cells. Count each
multi-cell colony as 1 transduced cell, as the cells will be dividing overthe 48 hour culture period (Figure 7).
The intensity of TurboGFP may vary between LentiORF
clones. If the TurboGFP intensity in your cells is low,
we recommend counting colonies at a higher magnification.

Figure 7. Example of individual colony
72 hour post-transduction. Four colonies
are circled. Image taken at 40x magnification.

15. Transducing units per mL (TU/mL) can be determined using thefollowing formula: # of TurboGFP positive colonies counted × dilution
factor × 40 = # TU/mL
25 μL of diluted virus was added to the cells.
This is 1/40th of a mL.

Example: 55 TurboGFP positive colonies counted in well A3. (TurboGFP
positive colonies) × 625 (dilution factor) × 40 = 1.38 × 106 TU/mL
106 can be expected from non-concentrated lentiviralparticles.

Table 4. Example of set up for dilutions

Well (Row A, B, C, or D)
Originating(96-well plate)Destination(24-well plate)

A1

Volume diluted
virus used

Dilution factor

25 μL

5*

A2

A1

25 μL

25

A3

A2

25 μL

125

A4

A3

25 μL

625

A5

A4

25 μL

3125

A6

A5

25 μL

15625

A7

A6

25 μL

78125

25 μL

390625 *A8horizondiscovery.com

Once you have generated a lentiviral stock with a suitable titer, you are readyto transduce the lentiviral vector into the mammalian cell line of choice andassay for expression of your recombinant protein.
Multiplicity of Infection (MOI)
To obtain optimal expression of your gene of interest, you will need totransduce the lentiviral vector into your mammalian cell line of choice using
a suitable MOI. MOI is defined as the number of transducing units per cell.
Although this is cell line dependent, it generally correlates with the numberof integration events per cell and, as a result, level of transgene expression.
Determining the optimal MOI
A number of factors can influence determination of an optimal MOI including
the nature of your mammalian cells (actively versus non-dividing), its relativetransduction efficiency, your application of interest, and the nature of yourgene of interest. If you are transducing your lentiviral construct into themammalian cell line of choice for the first time, after you have titered it,
we recommend using a range of MOIs (for example, 0, 0.5, 1, 2, 5, 10, 20) to
determine the MOI required to obtain optimal expression for your particularapplication. If minimal expression is preferred, it should be noted that toachieve single copy expression, an MOI of 0.3 is generally used followed by
antibiotic selection, which typically results in less than 4% of your cells having
more than one insert.

Protocol VIII - Transduction
Transduction of target cells
The protocol below is optimized for transduction of the lentiviral particles
into Dharmacon HEK293T, OVCAR8 or MCF7 cells in a 24-well plate using
serum-free medium. If a different culture dish is used, adjust the numberof cells, volumes and reagent quantities in proportion to the change insurface area (Table 5). It is strongly recommended that you optimize
transduction conditions to suit your target cell line for the highest
transduction efficiency possible.
It is preferable that transduction be carried out in medium that is serumfree and antibiotic-free. A reduction in transduction efficiency may occur
in the presence of serum. However, it is possible to carry out successfultransductions with serum present; you will need to optimize the protocol
according to your needs.
1. On day 0, plate 5 × 104 cells per well in a 24-well plate.
Incubate overnight.
2. The next day (day 1), remove the medium and add the virus to the MOI
you wish to use. Set up all desired experiments and controls in a similar
fashion. Bring the total volume of liquid up so that it just covers the cells
efficiently with serum-free medium (see Table 5 for guidelines).
If you are using concentrated virus you are likely to usevery little virus volume and a lot of serum-free medium; ifyou are using unconcentrated virus you will find you needmuch more virus volume.

3. Approximately 4-6 hours post-transduction, add 1 mL of complete
medium (serum plus pen-strep if you are using it) to your cells andincubate overnight.

Table 5. Suggested volumes of medium per surface area per well of adherent cells.

Surface area
per Well (cm2)

100 mm

56.0

5.0

60 mm

20.0

2.0

35 mm

8.0

1.0

6-well9.4

1.0

We have experienced low toxicity with transduction in thecell lines tested, therefore removal of virus is not required
for many cell lines. In our hands higher transduction
efficiencies have been achieved if the virus is not removed
after 6 hours. However, if toxicity is a problem, aspirate
the mixture after 4-6 hours and replace with fresh growth
medium. Additionally, fresh growth medium should be
replenished as required for continued cell growth.
At 48 hours post-transduction examine the cells microscopically for thepresence of reporter expression as this will be your first indication as tothe efficiency of your transduction (Figure 8).
When visualizing Evrogen TurboGFP expression, if lessthan 90% of all cells are green, it is recommended to utilize
Blasticidin S selection in order to increase the percentage
of cells expressing your gene of interest.
a. If adding Blasticidin S, use the appropriate concentration as
		 determined based on the kill curve experiment. If the cell density is		 close to or above 50% confluency, it is recommended that you split		 the cells at the time of Blasticidin S addition.
b. Approximately every 2–3 days replace with freshly prepared		selective medium.
c. Monitor the cells daily and observe the percentage of surviving		 cells. At some time point almost all of the cells surviving selection
		 will be harboring the gene of interest. Optimum effectiveness		 should be reached in 3–6 days with Blasticidin S, at which time all		 the cells surviving selection will harbor the gene of interest.
The higher the MOI that is used the more copies of thegene of interest and Blasticidin S resistance gene you will
have per cell. When selecting on Blasticidin S, it is worth
remembering that at higher MOIs, cells containing multiple
copies of the resistance gene can withstand higher
Blasticidin S concentrations than those at lower MOIs.
Even at single copy the marker in the pLOC vector provides
significant resistance to Blasticidin S in transduced cells.
Cells with very low transgene expression level may still
survive the selection even if TurboGFP is expressed at
an undetectable level (Figure 8). We recommend using
untransduced control cells to monitor the selection
process. Longer selection and higher concentrations ofBlasticidin S are recommended if high level of transgene
expression is desired. Adjust the concentration of
Blasticidin S to a level that will select for the populationof transduced cells you wish to select for, without going
below the minimum antibiotic concentration you haveestablished in your kill curve.

Suggested total
serum-free medium
volume per well (mL)

Tissue culture dish12-well

3.8

0.5

24-well

1.9

0.25

96-well

0.3

0.1

4. Once observed transduction efficiency is at an acceptable level (with orwithout Blasticidin S selection), you can proceed to assay cells for gene
expression by reverse transcription quantitative real-time PCR (RT-qPCR),
western blot analysis or other appropriate functional assay. The Evrogen
TurboRFP expression construct may be used as a control.
Optimal length of incubation from the start of transfection/transduction to analysis is dependent on cell type and thespecific gene being over expressed.

A

B

250 msec

4 sec

Figure 8. HEK293T cells transduced with Precision LentiORF expressing ANGPT1
HEK293T cells were plated at 200,000 cells/well (6-well format) and the next day
transduced at MOI 0.3. Two days after transduction, the cells were selected withBlasticidin S (2.5 µg/mL) for 5 days. A. Phase contrast. B. TurboGFP expression.

Protocol IX – determining relativetransduction efficiency
Follow the procedure below to determine the relative transduction efficiencyof purchased Dharmacon Precision LentiORF viral particles. This protocol
should be used with purchased Precision LentiORF RFP control in viralparticle format.
Prior to transducing with purchased Precision LentiORF individual clones inviral particle format, we recommend determining the relative transduction
efficiency of your cell type. Lentiviral titers provided with purchased Precision
LentiORF viral particles have been calculated by transducing Dharmacon
HEK293T cells. Transduction efficiencies vary significantly by cell type. The
relative transduction efficiency of your cells may be estimated by determining
the functional titer of a control virus such as the Precision LentiORF TurboRFP
control viral particles (Cat #OHS5833) in your cells of interest.
Follow the procedure below to determine the functional titer of the LentiORF
viral stock in the mammalian cell line of your choice. The following conditions
have been optimized for transduction of Dharmacon HEK293T cells. When
determining the relative transduction efficiency of your cell type, use thetransduction conditions that have been optimized for your cell type of choice.

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1.

The day before transduction, seed a 24-well tissue culture plate withyour cells at ~ 5 × 104 cells per well in their respective medium.
The following day, the well should be no more than
40-50% confluent.

2.

Make dilutions of the Dharmacon LentiORF control viral stock in around bottom 96-well plate using serum-free medium. Utilize theplate as shown in Figure 9 with one row for each replicate (werecommend performing at least two replicates). Use the procedure
below for dilution of the viral stock. The goal is to produce a series offive-fold dilutions to reach a final dilution of 390,625-fold (Figure 5).

1

2

3

4

5

6

Replicate 1 A
Replicate 2 B
Replicate 3 C
Replicate 4 D
Figure 9. A twenty-four well tissue culture plate seeded with desired cells is used totiter the virus.

3.

4.

5.

6.

7.

Add 40 µL of serum-free medium to each well in column 1. Add 80 µLof serum-free medium to each well of columns 2-8. If desired, include
8 μg/mL polybrene in the dilution medium.
Add 10 µL of thawed control virus stock to each well in column 1(five-fold dilution). Pipette contents of well up and down 10–15 times.
Discard pipette tip.
With new pipette tips, transfer 20 µL from each well of column 1 to thecorresponding well in column 2. Pipette contents of well up and down
10–15 times and discard pipette tips.
With new pipette tips, transfer 20 µL from each well of column 2 to thecorresponding well in column 3. Pipette contents of well up anddown 10–15 times and discard pipette tips.
Repeat transfers of 20 µL from columns 3 through 8, pipetting up
and down 10–15 times and changing pipette tips between eachdilution. Pre-incubate the dilutions of the virus stock for 5 minutes atroom temperature.
It is strongly recommended that you use a high quality
multichannel pipettor when performing multiple dilutions.

8.

Label the 24-well plate as shown in Figure 9 using one row foreach replicate.
9. Remove culture medium from the cells in the 24-well plate.
10. Add 225 µL of serum-free medium to each well.
11. Transduce cells by adding 25 μL of diluted control LentiORF lentivirus
from the original 96-well plate (Figure 5) to a well on the 24-welldestination plate (Figure 9) containing the cells.
For example, transfer 25 μL from well A2 of the 96-well plate into
well A1 in the 24-well plate (Table 4).
12. Incubate transduced cultures at 37 °C for 4–6 hours.
13. Add 1 mL of your medium (normal serum concentration).
14. Culture cells for 72 hours.

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15. Count the TurboGFP expressing cells or colonies of cells. Count each
multi-cell colony as 1 transduced cell, as the cells will be dividing
over the 72 hour culture period (Figure 7). Count the number ofTurboGFP expressing colonies in wells corresponding to at least
two viral dilutions.
The intensity of TurboGFP may vary between LentiORF
clones. If the TurboGFP intensity in your cells is low, we
recommend counting colonies at a higher magnification.
Transducing units per mL (TU/mL) can be determined using thefollowing formula:# of TurboGFP positive colonies counted × dilution
factor × 40 = # TU/mL
25 μL of diluted virus was added to the cells. This is
1/40th of a mL.
Example: 55 TurboGFP positive colonies counted in well A6.
55 (TurboGFP positive colonies) x 78,125 (dilution factor) x
40 = 1.72 x 108 TU/mL
16. The functional titer calculated for your cell line under your experimental
conditions can be used to determine the relative transduction efficiencyof your cell type by using the following formula:
Functional titer of Dharmacon Precision LentiORF RFP control
virus stock in your cell line
÷ Titer of LentiORF RFP control virus stock as calculated by
Dharmacon in HEK293T= Relative transduction efficiency
For example, if the titer of the LentiORF RFP control virus stock in HEK293T(as provided on the product specification sheet) is 6.9 x 108 TU/mL and thefunctional titer of the LentiORF RFP control virus stock in your cell line is
1.72 x 108 TU/mL, the relative transduction efficiency of your cell type is 0.25.
For accurate MOI calculations in your cells of interest, multiply the provided
titer for each construct by the relative transduction efficiency determined foryour cells of interest.
Once the relative transduction efficiency of the virus has been establishedin your cell line, use the optimized transduction conditions determined inProtocol VIII to transduce your cell line with the purchased Precision LentiORF
individual clones in viral particle format. If the titer of the control virus is notsatisfactory in your cell line you might consider choosing a different cell line
more permissive to transduction by lentivirus before proceeding.

Controls and validation
Precision LentiORF starter kits
The use of vector-based ORF expression is a powerful and versatile tool.
Successful gene expression in vitro is dependent on several variables including:1) The target cell line being studied, 2) Transfection and transduction
efficiency, 3) Abundance of the endogenous protein of interest in the target
cell line, and 4) Robust experimental protocols. For all these reasons it isvery important to run controlled experiments where the transfection andtransduction efficiencies are as high as possible and measurable.
Controls
The Precision LentiORF TurboRFP control has been validated for ORF
expression experiments (Figure 10). This control has been tested intransfection- and transduction-based experiments for TurboRFP
expression and selection in Blasticidin S.

AB

FAQS/troubleshootingC

For answers to questions that are not addressed here, please email technicalsupport at ts.dharmacon@horizondiscovery.com

20 msec

2 sec

250 msec

Figure 10. The HEK293T cells transduced with pLOC-TurboRFP lentiviralparticles. DharmaconHEK293T cells were plated at 7,000 cells/well in 200 μL of
medium (96-well format). The next day cells were transduced at an MOI of 20
in serum-free medium for 5 hours before full serum medium was added. Cells
were imaged at 72 hours after transduction (40x magnification).
A. Phase contrast. B. Nuclear TurboGFP expression. C. TurboRFP expression.

Protocol X – replication
Replication from individual glycerol stock
Prepare 2X LB broth as directed in Protocol I with 8% glycerol* (Fisher
Scientific Cat #BP2291) and the appropriate antibiotics, and inoculate fromglycerol stock. For archive replication, grow all Precision LentiORF clones at
37 °C in LB-Lennox (low salt) medium plus 100 μg/mL carbenicillin in order toprovide maximum stability of the clones.
Replication of plates
Prepare target plates by dispensing ~ 160 μL of 2x broth (low salt)
medium supplemented with 8% glycerol* and appropriate antibiotic(100 μg/mL carbenicillin).
Prepare source plates
1. Remove foil seals (Fisher Scientific Cat #12-565–475) while the source
plates are still frozen to minimize cross-contamination.
2. Thaw the source plates with the lid on. Wipe any condensation
underneath the lid with a paper wipe soaked in ethanol.
Replicate
1. Gently place a disposable replicator (Fisher Scientific Cat #NC9584102)
in the thawed source plate and lightly move the replicator around inside
the well to mix the culture. Make sure to scrape the bottom of the wells.
2. Gently remove the replicator from the source plate and gently place inthe target plate and mix in the same manner to transfer cells.
3. Dispose of the replicator.
4. Place the lids back on the source plates and target plates.
5. Repeat steps 1–4 until all plates have been replicated.
6. Seal and return the source plates to the -80 °C freezer.
7. Place the inoculated target plates in a 37 °C incubator for 18–19 hours.
Freeze at –80 °C for long term storage. Avoid long periods of storage
at room temperature or higher in order to control backgroundrecombination products.
Due to the tendency of viral vectors to recombine, we
recommend keeping the incubation times as short as
possible and avoid subculturing. Return to your glycerol
stock for each plasmid preparation.
*Glycerol can be omitted from the medium if you are culturing for plasmid
preparation. If making copies of the constructs for long term storage at –80 °C,
8% glycerol is required.

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What packaging system should I use for making lentivirus?
The pLOC vector is tat dependant, so you must use a packaging systemthat expresses the tat gene. For packaging our lentiviral constructs, werecommend the Trans-Lentiviral Packaging System. The Trans-Lentiviral ORF
Packaging System allows creation of a replication-incompetent2, HIV-1-based
lentivirus, which can be used to deliver and express your gene of interest
in either dividing or non- dividing mammalian cells. The Trans-Lentiviral
Packaging System uses a replication-incompetent lentivirus based on thetrans-lentiviral system developed by Kappes et al2. For protocols
and information on packaging pLOC with our Trans-Lentiviral Packaging
System, please see the product manual available on our website.
Can I use any 2nd generation packaging system with the pLOC vector?
The pLOC vector is tat-dependant, so you must use a packaging system that
expresses the tat gene.
What does the number 40 refer to in the formula for the calculationof titer?
The titer units are given in transducing units (TU) per mL, so the number 40 isused to convert the 25 μL used in the titration (“volume of diluted virus used,(Table 5) to one milliliter.
How can I make a stable cell line?
In order to generate stable cell lines, it is important to determine theminimum amount of Blasticidin S required to kill non-transfected/transduced
cells. This can be done by generating a Blasticidin S kill curve. After you havedetermined the appropriate concentration of Blasticidin S to use, you cantransfect or transduce your cells with the construct and culture with Blasticidin
S in order to select for those cells that have a stable integrant.
Where do you purchase Blasticidin S?
Blasticidin S is available from Fisher Scientific (Cat #BP2647–25).
If transduction into your cell line is unsuccessful, you may need toconsider the following list of factors influencing successful transduction:
1. Transduction efficiency is integrally related to the quality and thequantity of the virus you have produced. Factors to consider whentransducing include MOI (related to accurate titer), the presence of serumin the medium, the use of polybrene in the medium, length of exposure
to virus, and viral toxicity to your particular cells.
2. High quality transfer vector DNA and the appropriate and efficient viral
packaging are required to make high quality virus able to transduce
cells effectively.
3. All cell lines are not equally permissible to transduction by lentivirus.
You may consider testing additional cell lines to find one more suitablefor your experiments.

References
1. X. Wu, J.K. Wakefield, Development of a novel trans-lentiviral vector thataffords predictable safety. Mol. Ther. 2, 47–55 (2000).
2. J.C. Kappes, X. Wu, Safety considerations in vector development. Somat.
Cell Mol. Genet. 26, 147–158 (2001).

Label licenses
The shRNA and gene expression Products, use and applications, are coveredby pending and issued patents. Certain Label licenses govern the use of the
products, these can be found at dharmacon-licensing-statements. It is each
Buyer’s responsibility to determine which intellectual property rights heldby third parties may restrict the use of Products for a particular application.
Please review the Label Licenses governing all use of the shRNA and gene
expression Products.

To find the contact information in your country for your technology of interest, please visit us at
horizondiscovery.com/contact-us
Horizon Discovery, 8100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom©2020 The Horizon logo and other trademarks are the property of Horizon Discovery Limited, unless otherwise stated. DHARMACON, LENTIORF, and DHARMAFECT
are trademarks of Dharmacon Inc. TURBOGFP is a registered trademark of Evrogen. CLONASE and FASTDIGEST are trademarks of Thermo Fisher Scientific, Inc.

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