Download Scaffold design, function and over-expression of

Scaffold design, function and over-expression of lentiviral-based microRNAs
Angela Schoolmeesters, Melissa L. Kelley, Annaleen Vermeulen, Anja Smith, *Mayya Shveygert, *Xin Zhou, *Robert Blelloch
Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, USA
*
Eli and Edythe Broad Center of Regeneration Medicine and Stem Cell Research, Center for Reproductive Sciences and Department of Urology, University of California San Francisco, San Francisco, CA, USA
Efficient down-regulation of endogenous microRNA
targets on mRNA level and transduction of cells (GFP
expression) in primary, immune and neuronal cell lines
Selection of a functional, universal scaffold
MicroRNAs are short non-coding RNAs that regulate gene expression through targeted
degradation of one or more genes and have been shown to play an important role in
development, differentiation and disease. For example, microRNAs are critical for stem cell
function and differentiation. Also, dysregulation of microRNA expression has been associated
with the development and progression of many types of cancers. To better understand the
functional roles of microRNAs in phenotypes of interest, gain-of-function experiments can be
performed by introducing microRNA mimics into cells, which mirror the effects of endogenous
microRNAs. Studies with synthetic microRNA mimics are useful in easy-to-transfect cell types
as well as instances where phenotypes are readily observed over short time periods. However,
additional delivery strategies are required for cells that are refractory to transfection and for
phenotypes that develop over longer time points. Here we describe the strategy for scaffold
design and highlight the importance of choosing an optimal promoter in cells of interest for
the over-expression of lentiviral-based microRNA mimics. We demonstrate down-regulation
of gene targets in difficult-to-transfect primary, immune and neuronal cells (HuVEC, K562, SHSY5Y), and show mesenchymal to epithelial transition markers in MDA-MB-231 breast cancer
cells by over-expression of miR-429.
HUVEC
HUVEC
1.0
0.8
0.6
0.4
0.2
0.0
miR-486 miR-526 miR-374
miR-30
miR-26
miR miR-204 miR-126 Vector only Luc siRNA
SMARTvector
Normalized relative to controls (RLU)
Introduction
Optimized scaffold strategy
Approximates primary microRNA sequence
containing specific microRNA processing
requirements
Mature human microRNA sequence inserted
into a well-characterized primary microRNA
sequence (a universal scaffold; the shMIMIC
microRNA strategy)
Constructs are microRNA-specific and closer
to “endogenous” or “natural regulation”
• Native or non-native promoter
• Native genomic context
Suitable for examining individual microRNA
processing and regulation
More predictable expression and regulation
• Non-native promoter
• In non-native genomic context
Well-suited for functional analysis of
many microRNAs in multiple assays and
screening applications
5'
mature strand
passenger strand
0.4
miR-30
miR-26
Passenger Strand
0.60
0.40
PGK
UBC
1
2
3
4
5
6
7
8
9 10 11 12
Serial Dilutions
Serial Dilutions
TU (× 105) = 8.0
4.0
2.0
1.0
0.5
0.25
TU (× 105) = 8.0
4.0
2.0
1.0
0.5
TU (× 105) = 8.0
Empty
hCMV
mCMV
K()Ź
K()Ź
Promoter
hCMV
mCMV
P()Ź
P()Ź
CAG
CAG
PGK.
PGK.
UBC
UBC
A549
4.0
2.0
1.0
Empty
hCMV
K()Ź
MOI 20
MOI 5
MOI 20
0.5
Non-targeting
miR-B w/ miR-A HP and loop
miR-B
Maintaining natural secondary
structure improves function
ALDOA
K562
SH-SY5Y
Untreated
Negative Control
1.00
miR -429 (field 2)
miR -429 (field 1)
0.80
miR-122
SH-SY5Y
SH-SY5Y
1.8
1.6
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0.60
MOI 20 MOI 10 MOI 5 MOI 20 MOI 10 MOI 5 MOI 20 MOI 10 MOI 5
miR-429
Negative Control #4
Untreated
• Over-expression of miR-429 results in over-expression of E-cadherin and initiation of the
morphological change from mesenchymal to epithelial cells
• Down-regulation of ZEB1 (a putative miR-429 target) expression is also observed
0.40
0.20
0.00
Control Scaffold 1 Control Scaffold 2 Scaffold B - no 2° Scaffold B - with
2°
non-targeting
MDA-MB-231 cells were transduced in triplicate with either miR-429 shMIMIC microRNA or negative control (no
puromycin selection). Cells were fixed in 2% paraformaldehyde 96 hours post-transfection. Immunofluorescence was
used to visualize E-cadherin expression and cellular localization. E-cadherin protein localization is in red, while nuclei
stained with Hoechst 33342 are in blue. Silencing of ZEB1 expression (120 hours) was measured using RT-qPCR.
target mature
HeLa cells, co-transfection using 0.3 μL/well
Lipofectamine-2000 and 50 ng/well
psiCHECK-2 reporter plasmid + 50 ng/well
scaffold expressing plasmid. Measured
dual-luciferase assay at 48 hours.
TurboRFP intensity quantification
9.00E+03
MOI=1
MOI=2
8.00E+03
7.00E+03
6.00E+03
Conclusions
Preferred design strategy for expressing microRNAs
Optimized scaffold design derived from native human microRNAs
• Secondary structure retained to result in efficient processing and loading of the mature microRNA
• Scaffold selected to minimize star (*) strand or passenger strand processing and activity
Over-expression in neuronal, immune and primary cell lines possible
Research tool for long-term studies of microRNA function and creation of stable cell lines
shMIMIC microRNA features
• Lentiviral delivery results in stable integration of microRNA into the host cell genome
• Provided as cost- and time-saving lentiviral particles for direct transduction
• shMIMIC designs created for human, mouse and rat microRNAs in miRBase
• TurboGFP or TurboRFP (Evrogen) expression allows visual tracking of shMIMIC
microRNA expression
• Puromycin-selectable for creation and maintenance of stable cell lines
• Pseudotyped with broadly trophic VSVg envelope
• Multiple promoter options for optimal expression in your cells of interest
5.00E+03
4.00E+03
hCMV
mCMV
hEF1Ÿ
mEF1Ÿ
PGK
CAG
UBC
SMARTchoice
promoters
3.00E+03
2.00E+03
5’ LTR
1.00E+00
Ȍ
RRE
or
or
0.00E+00
mCMV
hCMV
-1.00E+03
mEF1α
hEF1α
UBC
Promoters
DAPI
TurboRFP
mCMV
hCMV
P()Ź
K()Ź
UBC
PGK
None
WPRE 3’ SIN LTR
SMARTvector universal scaffold
TRE3G
5' LTR
Ȍ
tGFP
RRE
or
tRFP
none
mCMV
PGK
K()Ÿ
P()Ÿ
SMARTchoice
promoters
PuroR 2A Tet-On 3G WPRE 3' SIN LTR
SMARTvector
universal scaffold
gelifesciences.com/dharmacon
5HSUHVHQWDWLYHLPDJHVVKRZLQJRSWLPDOSURPRWHUP()Ź
TurboRFP
tGFP IRES PuroR
tRFP IRES
PGK
DAPI
0.25
TM
UBC
Jurkat
MOI 5
ZEB1
P()Ź
CAG
MOI 20
miR-429
HUVEC
1.20
PGK.
HEK293T
MOI 5
Let-7a
HMGA2
Cell line transfected
Serial Dilutions
0.25
mCMV
Promoter
Promoter
Empty
0%
microRNA over-expressed
Target gene measured
or
C
D
E
F
G
H
CAG
30%
Gregory et al. Nat. Cell Biology 2008
Korpal et al. Biol. Chem 2008
Park et al. Genes & Development 2008
7KHPRXVH()ŹSURPRWHULVPRVWDFWLYH
in MEL-1 human embryonic stem cells
SMARTchoice Promoter Selection Plate
• Arrayed SMARTvector lentiviral particles; one row
for each promoter
• Wells contain SMARTvector Non-targeting control
expressing TurboGFP™ (Evrogen, Moscow, Russia)
• 2-fold dilutions of particles (TU), in duplicate
A
B
mEF1Ź
K562
K562
40%
10%
hLuc siRNA
8.0 4.0 2.0 1.0 0.5 0.25 8.0 4.0 2.0 1.0 0.5 0.25
TU (× 105) =
Promoter
hEF1Ź
miR-B w/ miR-A HP
miR-A w/ miR-B HP and loop
miR-A HP and loop
0.00
miR-A w/ miR-B HP
0.20
Vector only
MEL-1 cells were fixed and stained with anti-TurboRFP antibody and DAPI 72 hours posttransduction. Fluorescent images were collected on the GE IN Cell Analyzer 2000 system and
quantified using IN Cell Investigator High-content image analysis software.
Serial
Dilutions
Serial
Dilutions
mCMV
miR-126
Normalized Luciferase Expression
0.80
Choose the most active promoter in your
cells for best experimental results
hCMV
miR-204
1.00
3'
empty
miRSMARTvector
The flanking region contributes to function
Cytoplasmic TurboRFP Intesity/Nuclear DAPI Counts
Foreign sequence
50%
20%
0.2
Mature/Guide Strand
microRNA
scaffold
60%
MET phenotype induced by transduction of
hsa-miR-429 shMIMIC microRNA
0.6
1.20
1. Must be processed efficiently and consistently
2. Must accept foreign sequences & maintain
accurate processing
3. Must have good functionality from only
mature strand
4. Must have minimal passenger strand activity
(if passenger strand functional, this could
complicate your results)
70%
0.8
What is the best strategy for microRNA
over-expression using lentiviral particles?
microRNA primary transcript strategy
80%
1.0
0.0
Normalized Luciferase Expression
Borrowing the endogenous
pathway that processes host
encoded microRNA transcripts to
over-express functional mature
pri-microRNA
Nucleus
DNA
(3)
RNA
Encoded
microRNA. (1) The lentiviral particle
microRNA
(1)
Drosha
binds to the host cell and delivers
(4)
its engineered RNA genome, which includes
pre-microRNA
the encoded microRNA, to the cytoplasm. (2)
Exportin-5
(5)
The lentiviral genome is reverse-transcribed
Cytoplasm
Reverse
in the cytoplasm (i.e., RNA to DNA). The DNA
Transcription
Dicer
(2)
intermediate form is imported into the nucleus
(6)
Mature
and is stably integrated into the host genome.
microRNA Duplex
mRNA
(3) Dharmacon™ shMIMIC™ microRNAs are
destabilization
& protein
transcribed in the same manner as endogenous
down-regulation
Mature microRNA (7)
microRNA genes. Native and over-expressed priRISC
microRNA transcripts enter the microRNA processing
pathway, are processed by the Microprocessor complex
(4), shuttled out of the nucleus into the cytoplasm (5) and further
processed by the Dicer complex into active, mature microRNA sequences (6) which are incorporated
into the RNA Induced Silencing Complex (RISC). These microRNA-loaded RISC bind to target mRNAs and
cause transcript destabilization, resulting in down-regulation of protein expression (7).
90%
Cells were transduced with specific shMIMIC microRNA or Non-targeting Control
lentiviral particles (no puromycin selection). shMIMIC microRNA repression of specific
endogenous targets were determined using RT-qPCR at 120 hours post-transduction.
Data was normalized to a reference gene PPIB and further normalized to matched
SMARTvector Non-targeting Control.
Identification of a scaffold that promotes loading of the mature strand
1.2
100%
% Relative ZEB1 mRNA Expression
Normalized to Matched Negative Control
Normalized relative to controls (RLU)
Different microRNA scaffolds produce different levels of functionality
1.2
% Relative Target Expression
Normalized to Matched Negative Control
Abstract
All images at MOI = 2