Scaffold design, function and over-expression of lentiviral-based microRNAsAngela 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

TM

gelifesciences.com/dharmacon

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, SH-SY5Y), and show mesenchymal to epithelial transition markers in MDA-MB-231 breast cancer cells by over-expression of miR-429.

Borrowing the endogenous pathway that processes host encoded microRNA transcripts to over-express functional mature microRNA. (1) The lentiviral particle binds to the host cell and delivers its engineered RNA genome, which includes the encoded microRNA, to the cytoplasm. (2) The lentiviral genome is reverse-transcribed in the cytoplasm (i.e., RNA to DNA). The DNA intermediate form is imported into the nucleus and is stably integrated into the host genome. (3) Dharmacon™ shMIMIC™ microRNAs are transcribed in the same manner as endogenous microRNA genes. Native and over-expressed pri-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).

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.

microRNA primary transcript strategyApproximates primary microRNA sequence containing specific microRNA processing requirementsConstructs 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

Optimized scaffold strategyMature human microRNA sequence inserted into a well-characterized primary microRNA sequence (a universal scaffold; the shMIMIC microRNA strategy)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

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

• 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 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.

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 linesshMIMIC 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

Abstract

Introduction

What is the best strategy for microRNA over-expression using lentiviral particles?

The mouse EF1α promoter is most active in MEL-1 human embryonic stem cells

Efficient down-regulation of endogenous microRNA targets on mRNA level and transduction of cells (GFP expression) in primary, immune and neuronal cell lines

Conclusions

Selection of a functional, universal scaffold

MET phenotype induced by transduction of hsa-miR-429 shMIMIC microRNA

1. Must be processed efficiently and consistently2. Must accept foreign sequences & maintain

accurate processing3. Must have good functionality from only

mature strand4. Must have minimal passenger strand activity

(if passenger strand functional, this could complicate your results)

5’ LTR 3’ SIN LTR RRE WPRE

SMARTvector universal scaffold

PuroR

or

or

tGFP IRESIREStRFP

None

hCMV

mCMV

hEF1α

mEF1α

PGK CAG UBC

SMARTchoice promoters

Ψ

or

or

TRE3G

SMARTchoicepromoters

5' LTR Ψ tGFP

tRFP

none

WPRE 3' SIN LTR

SMARTvector universal scaffold

RRE Tet-On 3G

mCMVPGKhEF1αmEF1α

PuroR 2A

MEL-1 cells were fixed and stained with anti-TurboRFP antibody and DAPI 72 hours post-transduction. Fluorescent images were collected on the GE IN Cell Analyzer 2000 system and quantified using IN Cell Investigator High-content image analysis software.

TurboRFP intensity quantification

Cyto

plas

mic

Tur

boRF

P In

tesi

ty/N

ucle

ar D

API C

ount

s

-1.00E+03

0.00E+00

1.00E+00

2.00E+03

3.00E+03

4.00E+03

5.00E+03

6.00E+03

7.00E+03

8.00E+03

9.00E+03

mCMV hCMV mEF1α

Promoters

hEF1α UBC

MOI=1

PGK

MOI=2

Identification of a scaffold that promotes loading of the mature strand

The flanking region contributes to function Maintaining natural secondary structure improves function

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.

Different microRNA scaffolds produce different levels of functionality

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mature strandpassenger strand

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miR -429 (field 1) miR -429 (field 2)

Untreated Negative Control

miR -429 (field 1) miR -429 (field 2)

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miR-429 Negative Control #4 Untreated

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Gregory et al. Nat. Cell Biology 2008Korpal et al. Biol. Chem 2008Park et al. Genes & Development 2008

Mature/Guide Strand

Passenger Strand

5'

3'

microRNAscaffold

Foreign sequence

Nucleus

Drosha(4)

pri-microRNA(3)

ReverseTranscription

(2)

Mature microRNA Duplex

Mature microRNA (7)

pre-microRNA

Cytoplasm

RNAEncodedmicroRNA

(1)

DNA

Exportin-5(5)

Dicer(6)

mRNA destabilization

& proteindown-regulation

RISC

SH-SY5YHUVEC K562 SH-SY5YHUVEC K562

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Let-7a microRNA over-expressed miR-429 miR-122

HMGA2Target gene measured ZEB1 ALDOA

HUVECCell line transfected K562 SH-SY5Y

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mal

ized

to M

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ed N

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Let-7a miR over-expressed miR-429 miR-122

HMGA2Target gene measured ZEB1 ALDOA

HUVECCell line transfected K562 SH-SY5Y

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Let-7a miR over-expressed miR-429 miR-122

HMGA2Target gene measured ZEB1 ALDOA

HUVECCell line transfected K562 SH-SY5Y

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TU (× 105) = Promoter

Serial Dilutions

A

B

CDE

FGH

1 2 3 4 5 6 7 8 9 10 11 12

mCMV

mEF1α

hEF1α

CAG

PGK

UBC

hCMV

4.08.0 2.0 1.0 0.5 0.25 4.08.0 2.0 1.0 0.5 0.25

SH-SY5YHUVEC K562 SH-SY5YHUVEC K562

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Let-7a miR over-expressed miR-429 miR-122

HMGA2Target gene measured ZEB1 ALDOA

HUVECCell line transfected K562 SH-SY5Y

% R

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TurboRFP DAPI

mCMV

mEF1α

UBC

hCMV

hEF1α

PGK

All images at MOI = 2

TurboRFP DAPI

Representative images showing optimal promoter (mEF1α)

Choose the most active promoter in your cells for best experimental results

TU (× 105) =

A549

Serial Dilutions

8.0 4.0 2.0 1.0 0.5 0.25

Empty

hCMV

mCMV

hEF1α

mEF1α

CAG

PGK.

UBC

Prom

oter

TU (× 105) =

HEK293T

Prom

oter

Serial Dilutions

8.0 4.0 2.0 1.0 0.5 0.25

Empty

hCMV

mCMV

hEF1α

mEF1α

CAG

PGK.

UBC

TU (× 105) =

Jurkat

Prom

oter

Serial Dilutions

8.0 4.0 2.0 1.0 0.5 0.25

Empty

hCMV

mCMV

hEF1α

mEF1α

CAG

PGK.

UBC

Serial Dilutions

Identification of a scaffold that promotes loading of the mature strand

The flanking region contributes to function Maintaining natural secondary structure improves function

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.

Different microRNA scaffolds produce different levels of functionality

0.00

0.20

0.40

0.60

0.80

1.00

1.20

miR

-A H

P an

d lo

op

miR

-A w

/ m

iR-B

HP

miR

-A w

/ m

iR-B

HP

and

loop

miR

-B

miR

-B w

/ m

iR-A

HP

miR

-B w

/ m

iR-A

HP

and

loop

Non

-tar

getin

g

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Control Scaffold 1 Control Scaffold 2 Scaffold B - no 2° Scaffold B - with2°

non-targeting target mature

Nor

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ized

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ifera

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ized

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ifera

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miR-486 miR-526 miR-374 miR-30 miR-26 miR-204 miR-126 Vector only Luc siRNA

Nor

mal

ized

rel

ativ

e to

con

trol

s (R

LU)

miR-SMARTvector

Nor

mal

ized

rel

ativ

e to

con

trol

s (R

LU)

miR-30 miR-26 miR-SMARTvector

miR-204 miR-126 Vector only hLuc siRNA

mature strandpassenger strand

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