3’-UTR Luc/GFP Reporters

3’-UTR Luciferase Reporter Vectors MT-h00001-MT-h49999

3’-UTR Luciferase Reporter Viruses MV-h00001-MV-h49999
3’-UTR GFP Reporter Vectors MT-h50001-MT-h99999
3’-UTR GFP Reporter Viruses MV-h50001-MV-h49999
www.abmGood.co m

www.abmGood.co m
Table of Contents

Notice to the Purchaser 1

Technical Support 1

Biosafety 2

3’-UTR Reporter Clones 4

Protocol 5
Assay for Endogenously Expressed miRNAs with Transfection of Target 3’-UTR 5

Assay for Exogenously Expressed miRNA and Target 3’-UTR Interactions 6

Generation of 3’-UTR Cell Lines 7

Frequently Asked Questions 8

References 9

Contact Information 10

Notes 11

3’-UTR Luc/GFP Reporter Handbook

Notice to the Purchaser
The products are for research use only. Use in and/or for diagnostics or
therapeutics is strictly prohibited. By opening and using the product, the
purchaser agrees to the following: The components in this kit may not be
distributed, resold, or modified for resale without prior written approval from
Applied Biological Materials (abm) Inc. The information in this document
is subject to change without notice and should not be construed as a
commitment by abm Inc. or its affiliated corporations. In no event shall
abm Inc. or its affiliated corporations be liable for incidental or consequential
damages in connection with or arising from the use of this manual and
product.

abm Inc. products are warranted to meet our QC testing standards at the
time of shipment. Notice of problematic products must be made to abm
Inc. within 30 days of receipt of the product. This product warranty limits
abm Inc.’s liability to the replacement of the product only.

Technical Support
For more information on abm Inc. products, please visit our website:

http://www.abmGood.com

For additional information or technical assistance, please call or email us at:

Applied Biological Materials, Inc.

Phone: (604) 247-2416
1-866-757-2414
Fax: (604) 247-2414
E-mail: technical@abmGood.com

Page 1 of 11 3’-UTR Luc/GFP Reporter Handbook

Biosafety
Our 3’-UTR Lentivirus Reporter System employs 3rd generation self-inactivating
recombinant lentiviral vectors with enhanced biosafety and minimal relation
to the wild-type, human HIV-1 virus. The lentiviral particles produced with this
system are replication-incompetent and designed with a number of safety
features to enhance its biosafety. All Lentiviral Reporter Systems provided by
abm Inc. include the following safety features:
• An enhancer deletion in the U3 region of 3’ΔLTR ensures self-inactivation
of the lentiviral vector following transduction & integration into the target
cell’s genomic DNA.
• Utilization of a RSV promoter upstream of 5’ΔLTR allows efficient Tet-
independent production of viral RNA.
• The number of lentiviral genes necessary for packaging, replication and
transduction is limited to three (Gag/Pol/Rev), and their expression is derived
from different plasmids, all lacking packaging signals. The plasmids share
no significant homology to the expression vector, preventing the generation
of replication-competent virus.
• None of the Gag, Pol, or Rev genes will be present in the packaged viral
genome, thus making the mature virus replication-incompetent.

Despite the safety features discussed above, it is highly recommended

that all manipulation with lentiviral vectors, including viral production and
transduction, be performed under Biosafety Level 2 (BL-2). All published BL-2
guidelines with proper waste decontamination should be strictly followed. In
addition, exercise extra caution when creating lentivirus carrying potentially
harmful or toxic genes (e.g. oncogenes). For more information about the
BL-2 guidelines and lentivirus handling, refer to “Biosafety in Microbiological
and Biomedical Laboratories,” 4th Edition. This may be downloaded at:
http://www.cdc.gov/od/ohs/biosfty/bmbl4/bmbl4toc.htm

It is also important to consult with the health and safety officer(s) at your
institution for guidelines regarding the use of lentiviruses, and to always
follow standard microbiological practices, which include:
• Wearing gloves and a lab coat at all times.
• Always work with pseudoviral particles in a Class II culture facility and
ensure all procedures are performed carefully to minimize spillages.
• Work surfaces are thoroughly decontaminated daily and also immediately
after any spills of viable material.
• All cultures, stocks and other regulated wastes are decontaminated before
disposal by an approved decontamination method, such as autoclaving.

3’-UTR Luc/GFP Reporter Handbook Page 2 of 11

 Luc or GFP Luc or GFP
CMV Prom. CMV Prom.
Reporter 3’-UTR of Interest Reporter 3’-UTR of Interest

Reporter mRNA 3’-UTR Reporter mRNA 3’-UTR

target site and miRNA. target site and miRNA.

miRNA
Interaction between RISC Complex
No interaction between
miRNA-RISC Complex & miRNA miRNA-RISC Complex &
mRNA 3’-UTR region RISC Complex mRNA 3’-UTR region

Luc or GFP mRNA 3’-UTR of Interest Luc or GFP mRNA 3’-UTR of Interest

Translation Normal
Inhibited Translation

No Reporter Reporter
Expression Expression
Figure 1: Methodology behind target gene 3’-UTR reporters in validating specific miRNA
interactions. miRNAs that specifically bind to the 3’-UTR region of a target gene mRNA
transcript will inhibit the downstream translation of Luc/GFP reporter mRNA into protein.

Figure 2: Map of pLenti-Luc-UTR. The 3’-UTR of the gene of interest can be cloned into
the MCS. pLenti-GFP-UTR is identical except for replacement of Luc with GFP.

Page 3 of 11 3’-UTR Luc/GFP Reporter Handbook

 3’-UTR Reporter Vectors

MicroRNAs (miRNAs) are critical regulators for target mRNA stability and
translation (Bartel, DP. 2004 and 2009). Interactions among miRNAs and
their target mRNA transcripts are still relatively unknown for most genes.
Though computational prediction tools are helpful, a definitive relation has
to be determined by experimental assays. 3’-Luc/GFP-UTR reporters have
been shown by many researchers to validate these interactions between
miRNAs and target gene 3’-UTR regions via standard methods (Lai, EC. 2002;
Petersen, C.P. et al 2006).

abm’s 3’-UTR Luciferase/GFP reporter vectors are offered as ready-to-use

DNA plasmids for either transient (transfection) or stable (lentivirus infection)
expression assays. These plasmids can be used to assess the functions of the
miRNA in any cell type by transducing a Luc/GFP-UTR reporter construct into
your cells of interest. More often, an assay is performed by co-transfection
of a 3’-UTR reporter construct and a miRNA over-expression construct
(Figure 1). Any interaction between the over-expressed miRNA and its target
can be evaluated by comparing reporter activities from the co-transfected
samples against the samples transfected with a blank control vector
(lacking the 3’-UTR insert).

Each 3’-UTR sequence is amplified by PCR using a high fidelity Taq polymerase
based on the listed NCBI reference sequence provided. The amplified 3’-
UTR fragment is then sub-cloned into our pLenti-Luc/GFP-UTR backbone,
downstream of the luciferase or GFP stop codon. The final constructs then
undergo sequencing QC, to ensure the accuracy of the 3’-UTR insert.

All 3’-UTR constructs available from abm Inc. can also be packaged
into lentiviral particles for stable transduction (Figure 2). With lentiviral
particles, difficult-to-transfect cells, such as primary cells, can be efficiently
transduced for stable, long term expression of a 3’-UTR reporter.

3’-UTR Luc/GFP Reporter Handbook Page 4 of 11

 Protocol
A. Assay for Endogenous miRNA Expression With Transient
Transfection of 3’-UTR Reporter
1. DNA seed stock should first be transformed and amplified in an
authentic DH5a bacteria strain and selected with Kanamycin. It is
advisable to perform a maxi preparation to obtain enough DNA for
future applications.

2. One day before transfection, seed 293 cells at a density of 70% in a 6

well plate.

3. The following day, set up the transfection using a standard

transfection protocol (Refer to abm Cat. No. G2100 for protocol).

Transfection Control 1 Control 2 Experiment

pLenti-Luc/GFP Control + - -
pLenti-Luc/GFP-UTR - + +
pLenti-miRNA-Sponge - + -

5. Further validation should be carried out using the outlined set up

below. Note: The interactions between an miRNA and 3’-UTR can be
further validated with mutations within the 3’-UTR sequence. When co-
transfected with an miRNA sponge plasmid, an attenuated or abolished
interaction between the miRNA and the 3’-UTR should be observed.
Ensure that the difference in amount of DNA in each case is
compensated with a common plasmid, such as a pShuttle (abm
Cat. No. A001) to normalize the amount trasnfected between your
experiments.

Transfection Control Exp. 1 Exp. 2 Exp. 3 Exp. 4

pLenti-Luc/GFP Control + - - - -
pLenti-Luc/GFP-UTR - + - + -
pLenti-Luc/GFP-UTR Mutant - - + - +
pLenti-miRNA-Sponge - - - + +

6. Follow the transfection protocol provided with the transfection

reagent kit. For reporter and microRNA construct co-transfection,
adjust the ratio to 2:1 (miRNA Vector: 3’-UTR Reporter Vector).

7. 48-72 hours after transfection, assay reporter expression using

fluorescent microscopy or a luciferase assay kit (abm Cat. No. G287).

Page 5 of 11 3’-UTR Luc/GFP Reporter Handbook

Protocol
B. Assay for Exogenously Expressed miRNA and Target 3’-UTR
Interactions With Transient Transfection
To assay for interactions between an exogenously expressed miRNA and a 3’-
UTR Luc/GFP Reporter construct, we suggest setting up a screening experiment
using abm’s pLenti-miRNA vectors and pLenti-Luc/GFP-UTR vectors. The same
amount and quality of DNA should be used for each transfection. An RFP control
plasmid can be used for normalizing the variation between transfections.

1. Prepare the DNA and set up the transfection as indicated in steps 1-2
in Section A.

2. An experimental design shown in the table below can be used a

general guide:

Transfection Experiment Control

pLenti-Luc/GFP-UTR + +
pLenti-miRNA + -
pLenti-miRNA (Blank Control) - +

3. When positive interactions are identified, further validation should be

carried out in the following configurations. Be sure to compensate for
any difference in the amount of DNA used with a common plasmid
(such as a pShuttle).
Transfection Exp. 1 Exp. 2 Control
pLenti-Luc/GFP-UTR + - +
pLenti-Luc/GFP-UTR Mutant - + -
pLenti-miRNA + + -
pLenti-miRNA (Control) - - +

4. Follow the transfection protocol included in the transfection

reagent kit. For co-transfections of 3’-UTR Reporter and Lenti-miRNA
constructs, adjust the DNA ratio to 2:1 (pLenti-miRNA : 3’-UTR Luc/GFP
Reporter).

5. 48-72 hours later, assay reporter expression using either

fluorescent microscopy or a luciferase assay kit (abm cat. No. G287).
Results: Any suppression of the reporter expression when using
pLenti-miRNA with a mutant 3’-UTR Luc/GFP reporter validates a
specific interaction between the miRNA and 3’-UTR of the gene of
interest.

3’-UTR Luc/GFP Reporter Handbook Page 6 of 11

Protocol
C. Generation of 3’-UTR Cell Lines
An added capability with abm’s pLenti-miRNA and pLenti-Luc/GFP-UTR
constructs is generating stable cell lines through lentiviral transduction, to
enable expression of a specific 3’-UTR or miRNA. This is particularly useful
for difficult-to-transfect primary cells. With a stable 3’UTR GFP/Luc reporter
expressing cell line, a library of miRNAs can easily be screened by transfection
or lentivirus infection methods.

1. Subculture the target cells in a 6-well plate to a 70% density. Determine

an optimal killing curve for the cells by adding Puromycin to the
growth medium a range of concentrations between 0.1 and 1 µg/
ml.

2. Generate a high titre lentivirus using the chosen construct (refer to

Lentivirus Packaging Protocol http://www.abmgood.com/Adenovirus-
Lentivirus-Antibody-Stem-Cells-shRNA.html and transduce the
target cells by following the detailed protocol as outlined in the
provided Lentivirus Infection Protocol http://www.abmgood.
com/Adenovirus-Lentivirus-Antibody-Stem-Cells-shRNA.html.

3. Subculture cells from a 6 well plate to two or three 10 cm dishes at

different cell densities for stable clone selection by adding the optimal
concentration of puromycin as determined by the killing curve assay
from step 1.

4. It is critical to have a control condition carried out with the same

density of cells without lentivirus transduction, but still with puromycin
selection. Complete cell death in the control dish compared
with cell survival in transduced conditions indicates a successful
selection.

5. Once selection is completed, optimal stable clones can be screened

by luciferase assay or GFP expression. Then the reporter cell lines can be

Page 7 of 11 3’-UTR Luc/GFP Reporter Handbook

Frequently Asked Questions
Why should I use abm 3’-UTR reporter vectors?
abm Inc. has the largest collection of human and mouse 3’-UTR vectors in a
ready-to-use lentiviral format, at the most competitive pricing available.

These constructs can be used either for transient transfection or stable

transduction (when packaged into lentiviral particles). With lentiviral particles,
difficult-to-transfect cells like primary cells can be efficiently transduced.

What are appropriate controls in a 3’-UTR reporter assay?

Based on available literature and in house testing, we believe that miRNA
sponges (or 5x tandem repeats of the mature miRNA reverse complementary
sequence, downstream of the luciferase or GFP stop codon in pLenti-Luc or
GFP) can be used as excellent positive controls to validate your results.

Do I have to mutate the 3’UTR sequence to show the specificity of

miRNA and its target interaction?
Yes, mutant sequences are the best way to validate that an interaction
between a pLenti-miRNA and pLenti-Luc/GFP-UTR is specific. abm Inc. offers
a custom Site Directed Mutatagenesis Service at the material cost (http://
www.abmgood.com/Site-Directed-Mutagenesis-Service.html).

Do you offer custom lentivirus packaging service?

Yes, abm Inc. is a market leader in the production of recombinant lentiviral
vectors and lentiviral vector packaging services. These are offered at
the most competitive prices with fast turn around times. For complete
information about our services, please visit: http://www.abmgood.com/
Custom-Lentivirus-Services.html.

What is your 3’-UTR vector Guarantee?

abm Inc. warrants that each vector contains the exact sequence based on
gene bank information referenced. At abm’s discretion, free replacement of
any non-conforming product can be arranged if notified within 30 days of
the product’s receipt. If you experience any difficulty with an abm product,
please contact our Technical Support Staff at 1-866-757-2414 (toll free) or
at 604-247-2416 for local customers. Alternatively, email us at technical@
abmGood.com for an immediate written response.

3’-UTR Luc/GFP Reporter Handbook Page 8 of 11

References

Bartel, DP (2009). “MicroRNAs: target recognition and regulatory functions”.

Cell 136 (2): 215–33.

Bartel DP (January 2004). “MicroRNAs: genomics, biogenesis, mechanism,

and function”. Cell 116 (2): 281–97.

Lai E.C. Micro RNAs are complementary to 3’UTR sequence motifs that
mediate negative post-transcriptional regulation. Nature Genetics 30, 363
- 364 (2002)

Petersen, C.P. et al. Short RNAs repress translation after initiation in

mammalian cells. Molecular Cell 21, 533–542 (2006)

Page 9 of 11 3’-UTR Luc/GFP Reporter Handbook

 Contact Information
Applied Biological Materials Inc.
Website: Email:
www.abmGood.com General Information:
info@abmGood.com
Phone: Order Products:
(8:30am-4:30pm PST M-F)
order@abmGood.com
Toll Free: 1-866-757-2414
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Fax: (604) 247-2414 (24Hr.) technical@abmGood.com
siRNA:
Address: siRNA@abmGood.com
Suite #8-13520 Crestwood Place
Business Development:
Richmond, BC
bd@abmGood.com
Canada V6V 2G2

Distributors
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3’-UTR Luc/GFP Reporter Handbook Page 10 of 11

Notes

Page 11 of 11 3’-UTR Luc/GFP Reporter Handbook