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destination lentiviral vector containing attR4 and attR2 recombi-
nation sites in a double BP and LR recombination catalyzed by
respective clonases with the participation of an intermediate donor
vector equipped with the attP1 and attP2 sites. Double recombi-
nation ensures correct assembly of the promoter and EGFP-
miRNA cassette in the destination vector. The recombination is a
one-step procedure that allows to avoid multiple subcloning oper-
ations. The destination vector is the only one with the ampicillin
resistance, so other plasmids participating in the recombination
will not support bacterial cell growth on ampicillin plates.
Furthermore, the original destination vector contains the ccdB
gene between the recombination sites, which is toxic for normal
Escherichia coli strains, and the vector will not support the cell
growth unless recombination is successful and ccdB gene is replaced
with the promoter-EGFP-miRNA construct. This is a clever
arrangement allowing for easy, fast, and fl exible manipulation of
miRNA clones as needed.
3
Procedures
3.1 Selecting miRNA
Sequence
The fi rst and critically important step in the miRNA knockdown
experiment is to select active miRNA sequences for the silencing of
your gene of interest. There are several ways to do that. Several
companies now offer lentiviral vectors encoding known endoge-
nous human, mouse, and, in some cases, rat miRNAs. These viruses
normally contain not only the endogenous hairpin structure but
also endogenous upstream and downstream regions. Presumably,
these miRNAs should be effi ciently processed by the cellular
machinery and can be used to knockdown genes they normally
regulate for experimental purposes. Alternatively, predesigned
miRNAs to many genes are offered commercially. Actually, the
part that is unique for these miRNAs is the sequence correspond-
ing to the stem in the miRNA hairpin complementary to the target
miRNA. Other elements, such as the loop and regions fl anking the
hairpin that are a part of the pre-miRNA, are usually standard
derived from an endogenous miRNA to ensure effi cient processing
of the “custom” miRNA in cells. For example, in the Invitrogen
system, fl anking sequences from murine mi-155 are used, which
are incorporated into the cloning vector. The miRNAs are sold as
single-stranded oligonucleotides encoding the stem and loop
sequences ready to be annealed and cloned into the cloning vector.
Finally, it is possible to design a custom miRNA sequence using a
miRNA designer software such as Invitrogen's BLOCK-iT™ RNAi
Designer. This is necessary when no predesigned miRNA is avail-
able for the gene or species of interest. This custom miRNA is then
cloned into the cloning vector the same way as predesigned
miRNA. Many companies also provide a negative control miRNA
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