Biology Reference
In-Depth Information
Chapter 15
Lentiviral Overexpression of miRNAs
Hannah Zöllner , Stephan A. Hahn , and Abdelouahid Maghnouj
Abstract
Deregulation of microRNAs (miRNAs) has been attributed to almost any human disease analyzed to date.
This calls for models and experimental strategies for functional analyses of miRNAs enabling miRNA over-
expression or suppression in target cell and/or tissues. Lentiviral vector (LV)-based technologies allow the
long-term overexpression of miRNAs in nearly all cell types in an easy and rapid manner and are therefore
popular tools for this application. In this chapter we describe the cloning of LV miRNA expression vectors
as well as the production of virus particles for target cell infection and stable expression of miRNAs.
Key words
MicroRNA overexpression, Lentiviral vector, Cloning, Sh-miR, Pri-miR
1
Introduction
To study the function of specifi c miRNAs, overexpression or
suppression techniques are commonly used to reach this goal. The
currently most widely used miRNA mimic (miR-Mimic) strategy
for miRNA overexpression is based on synthetic nonnatural
double-stranded miRNA-like RNA. This approach is limited to
transient expression of the miRNA under study. LV-based expres-
sion of miRNAs however allows the stable and long-term expres-
sion of an miRNA hairpin sequence in a large variety of cell types,
including those that are diffi cult to transfect with standard technol-
ogy such as primary or nondividing cells [
1
,
2
].
MiRNA expression cassettes in LV can be designed as small
hairpins (sh-miRs) or as primary miRNA (pri-miRs). Sh-miRs are
perfect hairpins with a small apical loop and a 3
′
UU-overhang.
The 3
end of the loop sequence contains the endogenous miR
sequence (herein called sense sequence,
see
also Fig.
1
) because it is
preferred for producing the mature miRNA sequence, whereas the
5
′
end of the loop sequence contains the fully complementary
sequence to the mature miRNA (herein called anti-mature
sequence) [
3
]. Sh-RNA synthesis systems are driven by RNA poly-
merase III (pol III) promoters such as variants of U6 or H1
′
