Biology Reference
In-Depth Information
Fig. 1
Map of pLKO.1-puro vector containing a sh-miR insert (here hsa-miR-30a-5p). The U6 promoter directs
RNA polymerase III transcription of the sh-miR. The sh-miR sequence is followed by a polyT termination signal
for RNA polymerase III.
Mature
indicates the position of the mature miRNA sequence under study.
Anti - mature
indicates the sequence 100 % complementary to the miRNA under study
promoters [
4
]. Among the main advantages of the sh-miR approach
is the ease of its cloning strategy which only requires the commer-
cial synthesis of two complementary oligonucleotides. Expressing
an miRNA as a pri-miR in turn requires the PCR amplifi cation of
a genomic fragment including the approximately 100 bp long
precursor-miRNA (pre-miR) sequence fl anked by some 200-
300 bp upstream and downstream of the pre-miR sequence. Apart
from decoupling the pri-miR expression from its endogenous pro-
moter, the expression and maturation of vector-based pri-miRs
include all endogenous processing steps and may thus better mimic
natural miRNA biogenesis. Furthermore, both the endogenous
so-called major and minor miRNAs are produced with this
