Biology Reference
In-Depth Information
expression vector system. Thus, if the experimental goal requires
the specifi c testing of only the minor or major miRNA sequence,
the expression of an miRNA-sponge vector competing out the
unwanted miRNA sequence may be entertained [
3
].
The LV system described herein is an HIV-based third genera-
tion three-vector system [
1
]. The fi rst vector contains the LTRs
(Long Terminal Repeats) required for reverse transcription and
integration, the packaging signal
and the heterologous sequences
(here miRNA sequence). The two remaining plasmids, the so-called
helper plasmids, which provide all the structural proteins (HIV-1
Gag/Pol, Tat, Rev, and Env), are needed in order to package the
new virus within the packaging cell [
5
]. Due to the
ψ
deletion
within the helper plasmids, this transcribed RNA does not get
incorporated into the new recombinant virus. Choosing the enve-
lope (Env) protein for pseudotyping and thus controlling the tro-
pism of the virus is important. The VSV-G protein (G glycoprotein
of the vesicular stomatitis virus) is often used because it allows the
transduction of a wide variety of cells [
4
]. For lentivirus production,
the packaging cell line HEK 293T is transiently transfected with the
three plasmids. Following transfer of the lentiviral particles gener-
ated by the HEK 293T cells to the target cells the VSV-G interact
with a phospholipid component of the cell membrane. This induces
the fusion of the membranes of the virus-envelope and the host
cell. After the entry of the virus, uncoating and reverse transcription
start immediately and subsequently the formation of a pre-integra-
tion complex (PIC) takes place. The PIC is a specifi c hallmark of
lentiviruses, enabling them to actively enter the nucleus of a cell in
order to infect nondividing cells. As soon as the viral DNA is inte-
grated into the host cell genome, gene expression is initiated [
6
].
ψ
2
Material
The overexpression of miRNAs via lentiviral particles requires
Biosafety Level 2 (BSL-2). Handle particles as a potentially biohaz-
ardous material and strictly follow all published BSL-2 guidelines.
The transduced target cells can be handled as Biosafety Level 1
after several round of medium exchange following removal of the
viral particles. Prepare all solutions using ultrapure water (18 M
Ω
cm at 25 °C) and sterile fi ltrate (0.2
m pore size); aliquots are
stored at −20 °C (unless indicated otherwise).
μ
1. For sh-miR expression: pLKO.1 puro (addgene no. 8453).
2. For pri-miR expression: pLJM1-EGFP (addgene no. 19319).
3. For HIV-1 GAG/POL, Tat and Rev expression (helper plas-
mid): pCMV delta R8.2 (addgene no. 12263).
4. For VSV-G expression (helper plasmid): pCMV-VSV-G (add-
gene no. 8454) or pHIT/G [
7
].
2.1 Suggested
Vectors
