Vector-Based Human microRNA Expression Clones and Target
Validation Expression Clones
MicroRNAs (or miRNAs) are small non-coding
single-stranded RNA molecules that are transcribed from the genomes of
eukaryotic organisms. They are highly conserved and usually 21~23
nucleotides in length. MiRNAs have been found to be involved in almost
all cellular activities and functions. They regulate the expression of
genes by binding to the 3’ untranslated regions (3’-UTRs) of targeted
mRNAs. Irregularities in miRNA regulated gene expression have been
found to be associated with cancers, cardiovascular disorders, and a
variety of other diseases.
GeneCopoeia is now offering:
more than 600 pre-made OmicsLink™ human Precursor miRNA expression
clones
miRNA target sequence expression clones that cover 20,000 human
genes
Researchers can use these miRNA expression clones to study the
expression regulation of corresponding genes/proteins. In combination
with target sequence (3’-UTR) expression clones, which have luciferase
as the reporter gene, researchers can also cross-validate.
GeneCopoeia also offers multiple sets of full length human and
mouse expression ready ORF cDNA clones that can be used for
gain-of-function studies of corresponding target genes.
OmicsLink™ Human Precursor miRNA Expression Clones
GeneCopoeia offers precursor miRNA expression clones constructed in
a feline immunodeficiency virus (FIV) based lentiviral vector system. A
RNA polymerase III type promoter (H1) is used to drive transcription
of hairpin precursor miRNAs (approximately 150 nucleotides), which are
then processed to generate mature miRNAs by the enzymes involved in
RNAi machinery. A tracking/reporter gene EGFP is co-expressed with the puromycin selection gene under the control of CMV promoter and bicistronic IRES element, which provides a
enient way for
monitoring miRNA expression, transfection and transduction efficiency.
Specific gene regulation is achieved as the result of binding of
RISC-miRNA complexes with the 3’UTR of the mRNAs of target genes (Fig.
1): 1) expression translation suppression and 2) mRNA cleavage,
degradation, and translation reduction.
Fig. 1. Lentiviral vector based precursor miRNA expression clones and the
mechanism of vector mediated miRNA gene regulation
Features of Precursor miRNA Expression Clone Collections
650 human precursor miRNA expression clones are available which
cover more than 95% of 678 known miRNAs in release 11.0 in miRBase
The expression cassettes of all miRNA expression clones have been
fully sequenced
Lentiviral vector based expression constructs allow efficient
infection based transduction of miRNAs into non-dividing and
difficult to transfect target cells as well as transfection based
transduction into conventional dividing cells
Stable cell line selection marker (puromycin) enables studies of
the effect of both long term and transient expression regulation
Vector plasmid transfection and virus infection transduction
efficiency are monitored by EGFP reporter protein.
OmicsLink™ Human miRNA Target Sequence (3’ UTR) Expression Clones
GeneCopoeia offers genome-wide miRNA target sequence (3’-UTR)
expression clone in a mammalian expression vector system. All 3’-UTR
sequences in these expression constructs are curated from public
domain gene sequence database. 3’-UTR Sequences immediately after
coding sequences are subsequently cloned after a reporter gene
(firefly luciferase) in a mammalian expression vector. The expression
of the reporter gene firefly luciferase is controlled by SV40
promoter. A tracking reporter gene, renilla luciferase, is also
constructed in the vector system, which provides tracking indicators
for successful transfection and expression of these miRNA target
sequence constructs in target cells. Figure 2 shows the basic features
of such an expression construct.
The regulatory effect of a particular miRNA on its potential target
sequences is assessed by the firefly luciferase functional assay.
These expression constructs transcribe a chimeric mRNA consisting of
coding ORF sequence for firefly luciferase and a 3’-UTR target
sequence. Upon binding of the 3’-UTR target sequence by a targeting
miRNA, enzymatic activity of the luciferase encoded by the firefly
luciferase ORF in the chimeric mRNA will be regulated accordingly. The
effect can be quantified by the colorimetric luciferase assay.
Figure 2. Vector features for human miRNA target sequence
expression clones
Applications of Precursor miRNA and Target Expression Clones
Gene expression regulation. The regulatory and functional effect
of the miRNAs can be studied individually or systematically using the
whole set or a subset of miRNA expression constructs by transduction
of precursor miRNAs into target cells either via transfection or
viral infection
MiRNA target gene identification and validation. Together with
GeneCopoeia’s miRNA target validation expression clones, miRNA
expression constructs can be used to identify and verify their target
genes
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