Exp. Anim. 66(1), 41–50, 2017

—Original—

RNAi-mediated knockdown of mouse melanocortin-4
receptor in vitro and in vivo, using an siRNA
expression construct based on the mir-187 precursor
Minoru Kato1), Yi-Ying Huang2), Mina Matsuo3, 8), Yoko Takashina4),
Kazuyo Sasaki4), Yasushi Horai5), Aya Juni6), Shin-Ichi Kamijo3, 9), Kaoru Saigo6),
Kumiko Ui-Tei7), and Hajime Tei2, 10)1) Research Unit/Neuroscience, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama,

Kanagawa 227-0033, Japan

2) Laboratory of Chronogenomics, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo

194-8511, Japan

3) Experimental Animal Laboratory, Mitsubishi Kagaku Institute of Life Science, 11 Minamiooya, Machida, Tokyo

194-8511, Japan

4) Pharmacology Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama,

Kanagawa 227-0033, Japan

5) Safety Research Laboratory, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama,

Kanagawa 227-0033, Japan

6) Department of Biophysics and Biochemistry, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi,

Bunkyo-ku, Tokyo 113-8657, Japan

7) Department of Biological Sciences, Graduate School of Science, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku,

Tokyo 113-8657, Japan

8) Present address: Division of Animal Experimental Laboratory, Life Science Research Center, University of Toyama,

2630 Sugitani, Toyama 930-0194, Japan

9) Present address: Division of Planning of Laboratory Animal Facility, Lifewill Corporation, 2740-12 Shimo-oyamada, Machida, Tokyo 194-0202, Japan

10)Present address: Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa,

Ishikawa 920-1192, Japan

Abstract: RNA interference (RNAi) is a powerful tool for the study of gene function in mammaliansystems, including transgenic mice. Here, we report a gene knockdown system based on the human
mir-187 precursor. We introduced small interfering RNA (siRNA) sequences against the mousemelanocortin-4 receptor (mMc4r) to alter the targeting of miR-187. The siRNA-expressing cassette
was placed under the control of the cytomegalovirus (CMV) early enhancer/chicken β-actin promoter.
In vitro, the construct efficiently knocked down the gene expression of a co-transfected mMc4rexpression vector in cultured mammalian cells. Using this construct, we generated a transgenic
mouse line which exhibited partial but significant knockdown of mMc4r mRNA in various brain regions.
Northern blot analysis detected transgenic expression of mMc4r siRNA in these regions. Furthermore,
the transgenic mice fed a normal diet ate 9% more and were 30% heavier than wild-type sibs. They
also developed hyperinsulinemia and fatty liver as do mMc4r knockout mice. We determined that thissiRNA expression construct based on mir-187 is a practical and useful tool for gene functional studies
in vitro as well as in vivo.
Key words: mir-187, mMC4R, RNAi, Transgenic mice
(Received 4 August 2016 / Accepted 15 September 2016 / Published online in J-STAGE 30 September 2016)
Address corresponding: M. Kato, Research Unit/Neuroscience, Mitsubishi Tanabe Pharma Corporation, 1000 Kamoshida, Aoba-ku, Yokohama,
Kanagawa 227-0033, Japan/H. TEI, Graduate School of Natural Science and Technology, Kanazawa University, Kakuma, Kanazawa, Ishikawa
920-1192, Japan
Supplementary Figure: refer to J-STAGE: https://www.jstage.jst.go.jp/browse/expanim©2017 Japanese Association for Laboratory Animal Science42

M. KATO, ET AL.

Introduction
RNA interference (RNAi) is a sequence-specific genesilencing mechanism widely conserved among eukaryotic cells [19]. RNAi is triggered in mammalian cells bysmall interfering RNAs (siRNAs) that are 21 nucleotides(nt) long and contain 2 nt 3′ overhangs.
Animal cells contain numerous endogenous ~22 nt
RNAs known as microRNAs (miRNAs) [16] that cancause mRNA degradation and/or translational inhibition
when bound to partially complementary sites in the 3′
untranslated region (3′-UTR) of mRNAs [32, 34]. Natural miRNAs are transcribed by RNA polymerase II (polII) as long, primary, 5′-capped, and polyadenylated miRNAs (pri-miRNAs) [3, 17]. The pri-miRNA is cropped
first to release a 70–90 nt hairpin-shaped precursor (premiRNA) by Drosha, a member of the ribonuclease III(RNase III) protein family, and its cofactor, DGCR8, inthe context of the microprocessor complex [6]. The premiRNA is recognized by the exportin-5-RanGTP heterodimer, and the pre-miRNA is exported to the cytoplasm [18, 33]. In the cytoplasm, pre-miRNA is cut by
the RNase III Dicer to generate a ~22 nt miRNA duplex[12, 14]. One strand of the Dicer product remains as amature miRNA and is assembled into an effector complex known as the miRNA-induced silencing complex(miRISC), which has a gene-silencing effect [15, 26].
The RNA pol II-driven short hairpin RNA (shRNA) expression system was constructed by replacing the miRNA
with siRNA.
RNAi has been used for the functional knockdown of
specific proteins in several experimental systems, from
cultured cells [7] to whole organisms, including mammals [25]. The RNAi technology represents an interesting tool in reverse genetic studies for several reasons.
First, RNAi usually results in partial inhibition of gene
expression and thus allows the generation of hypomorphic phenotypes, which are usually impossible to generateby complete knockout via gene targeting [9] or gene
editing technologies [31]. Second, the expression of
miRNA can be controlled using a large repertoire of RNA
pol II-dependent promoters [35], including temporally
and spatially specific and inducible promoters [20]. Consequently, the RNA pol II-mediated RNAi strategy canbe a powerful alternative to conventional gene knockout
technology.
miR-187, an miRNA that is highly expressed in neurons, has been intensively studied, and is considered apotential biomarker in the early diagnosis of a wide rangeof human cancers [8]. We chose to engineer mir-187
because DraIII and PshAI recognition sites in its precursor sequence allow for the easy replacement of its siRNA.
To confirm the knockdown efficacy of an endogenous
gene in vivo, we selected the mouse melanocortin-4 receptor (mMc4r) gene as the target. The mMC4R is aseven-transmembrane G protein-coupled receptor thatis predominantly expressed in the hypothalamic nuclei,
and is implicated in the regulation of food intake andbody weight [2]. Homozygous mMc4r knockout mice
are known to express an obese phenotype caused byexcess food intake. On the other hand, heterozygous
mMc4r knockout mice express an intermediate phenotype, indicating that obesity is tightly coupled to genedosage [11]. Thus the reduction of mMc4r expression inknockdown mice can be conveniently followed bymonitoring body weight.
To test the utility of an mir-187-based shRNA construct driven by an RNA pol II promoter, we used acytomegalovirus (CMV) early enhancer/chicken β-actin
promoter (CAG)-driven expression vector with the potential for expression in a range of tissues and cells [23].
Materials and Methods
Plasmids
An siRNA-expressing construct was generated as follows. The human miR-187 expression region, containing
the mir-187 stem-loop region and 5′ and 3′ flanking regions (271 bp in total), was PCR-amplified from HeLa
genomic DNA using the primers 5′-CGCGGATCCATCG G G AT G C A C A G C A A G T- 3 ′ a n d 5 ′ - G C T C TAGACCCACCAGAGCCTGGACTTTC-3′, digested
with BamHI/XbaI and ligated into the same restriction
sites in the pCAG expression vector (pCAG-miR-187).
Next, an EF1-α/EGFP expression cassette and an SV40/
blasticidin expression cassette were introduced to visualize the efficacy of transfection and to establish stable celllines (pGKD-miR-187). Hairpin sequences specific to thetargeted mRNAs were inserted into the DraIII/PshAI siteof either the pCAG-miR-187 or pGKD-miR-187 vector(pCAG-miR-mMC4R, pCAG-miR-Luc, and pGKD-miRmMC4R). The inserted oligonucleotide sequences wereas follows: mMC4R 5′-GTGACCCTCCAGGATGCTATGAG C AA C TTTTT T GTGAAG C C A C AGATG GAAAAAAGTTGCTCATAGCATCCTGAGGGAC-

RNAi CONSTRUCT BASED ON mir-187

43

Fig. 1.	Schematic structure of siRNA-expression cassette and constructs. A: siRNA-expressing cassette based on the mir187 precursor sequence; italic: mir-30 loop sequence, bold: siRNA sequence. B: Schematic structure of pCAGmiR-187 construct. C: Schematic structure of pGKD-miR-187 construct; arrowhead: NcoI restriction site, solid bar:probe for identification of transgenic mice.

GC-3′, luciferase: 5′-GTGACCCTCCAGATTTCGAGTCGTCTTAATGTTGTGAAGCCACAGATGGACATTAAGACGACTCGAAATCTGAGGGACGC-3′. Underlines indicate modified bases in the 5′ flanking and loop
regions. The construct pcDNA3.1-mMC4R contains themMC4R coding region isolated by PCR.
Cell culture and transfection
Human embryonic kidney-derived 293 (HEK293)
cells and HeLa cells were grown in DMEM (Thermo
Scientific; Logan, UT) supplemented with 10% fetal
bovine serum (FBS). Transfections were performed with
Lipofectamine Plus (Invitrogen; Carlsbad, CA) as directed by the manufacturer. Cells were co-transfectedwith pcDNA3.1-mMC4R vector and pCAG vectors at aratio of 5:1 (wt/wt).
Mice and ethics statement
We purchased C57BL/6J mice from the Charles River Laboratory (Yokohama, Japan). Mice were housed
under pathogen-free conditions. All procedures involving mice were performed in compliance with National
Institutes of Health guidelines and were approved by the
Animal Care and Use Committee of Mitsubishi Kagaku

Institute of Life Sciences, MITILS. Our animal husbandry abided by MITILS guidelines.
Generation and genotyping of miR-mMC4R transgenic
miceTransgenic mice were generated by microinjection
into C57BL/6J embryos of the pGKD-miR-mMC4R vector linearized by SalI digestion, using standard techniques [10], and a transgenic line was maintained bycrosses with C57BL/6J mice. Transgenic mice were
genotyped by Southern blot analysis of tail genomic
DNA. In Southern blots, genomic DNAs were digested
with NcoI, and a 32P-labelled EGFP fragment was used
as a probe (Fig. 1C). We used male mice in whole experiments in this study except the initial screening by
weight determination of founder transgenic mice. In
addition, we used non-transgenic male littermates ascontrol animals.
RNA extraction and qPCR
Total RNA and low-molecular-weight RNA-enrichedfractions were isolated using TRIZOL reagent (Invitrogen) or an mirVanan miRNA isolation kit (Ambion;
Austin, TX), following the manufacturers’ protocols.

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M. KATO, ET AL.

cDNAs were produced using SuperscriptII Reverse Transcriptase (Invitrogen). The relative expression of mMc4r
was estimated by qPCR using SYBR Premix Ex TaqII
reagents (TaKaRa; Kyoto, Japan) under standard reaction
conditions. The primer sequences were as follows: mouse
MC4R-F, 5′-CGCCAGGGTACCAACATGAAG-3′;
mouse MC4R-R, 5′-ACATGAAGCACACGCAGTATGG-3′; mouse β-actin-F, 5′-GGCCAACCGTGAAAAGATGA-3′; mouse β-actin-R, CACAGCCTGGATGGCTACGTA-3′. Primers for human GAPDH werepurchased from TaKaRa.

blood, liver, pancreas, and mesenteric fat were collected
and weighed. Plasma glucose concentrations were determined using a Glucose-CII Test Kit (Wako Pure
Chemical Industries, Osaka, Japan). Insulin concentrations were measured with an enzyme-linked immunosorbent assay (Morinaga Institute of Biologic Science,
Yokohama, Japan). Triglyceride levels were measured
using a Triglyceride E-test Kit (Wako). Plasma freefatty acid levels were measured using an NEFA C-test
Kit (Wako). Plasma leptin levels were measured using
Mouse Leptin ELISA kits (Morinaga).

Northern blotting for small RNA
In brief, 15% polyacrylamide-8M urea gels were used
to isolate the low-molecular-weight RNA-enriched fraction. Band sizes were estimated by comparing them toa 10 bp DNA Marker (NEB; Ipswich, MA). Gels weretransferred to Hybond-N+ membranes (GE Healthcare;
Uppsala, Sweden) by capillary transfer. After crosslinking, membranes were placed in DIG-easy Hyb(Roche; Indianapolis, IN). DIG-labeled riboprobes weresynthesized using an mirVana miRNA probe construction
kit (Ambion) using the following DNA oligonucleotide
as a template: miR-mMC4R sense probe (for guidestrand siRNA detection), 5′-AAAAAGTTGCTCATAGCATCCCCTGTCTC-3′. After hybridization at room
temperature overnight, membranes were washed twice
at 37°C for 15 min in 0.1 × SSC, 0.1% SDS. Probe detection was performed using a DIG Luminescent Detection
Kit (Roche) according to the manufacturer’s protocol.
Stripping of the riboprobe was performed at 68°C for
30 min in 0.1% SDS, followed by washing in 2× SSC atroom temperature. Then membranes were reprobed with
miR-16 riboprobe (Ambion) in the same manner as described above.

Statistical analysis
Statistical analysis was performed using unpaired twotailed Student’s t-tests.

Measurement of body weight and food consumption
Body weight was regularly measured, beginning at
3–4 weeks of age. Food intake was measured for maletransgenic and non-transgenic mice at 37 to 41 weeks ofage. A sufficient amount of food for a 9-day period wasweighed and provided to the mice ad libitum. Each weekday morning, the remaining food was weighed, for a
total of six measurements.
Determination of metabolic parameters
Fasting male transgenic and non-transgenic mice at
41 weeks of age were sacrificed under anesthesia, andResults
Assembly of an siRNA-expression construct based on
mir-187 precursor sequences
We constructed an siRNA-expressing cassette containing mir-187 precursor sequences with mir-30 loop sequences (Fig. 1A). Minor modifications to the flanking
and loop sequences were introduced to obtain the highest knockdown efficacy by using Dual-Luciferase Reporter Assay system (Fig. 2). In the original shLuciferase
cassette with no modification, the G:C pair at the neckof the mir-30 loop was changed to UG wobble pair toopen easily in modified siRNA-expressing cassette,
Modification (Mod) 1, and 4. As shown in our siRNAdesign algorithm (22, 29), the 5′ terminal residue offunctional siRNA guide strand is preferable to be A orU, and Dicer cleavage site is considered to be at two
nucleotides inside from the double strand terminal. Thus,
the relative luciferase activity is evidently reduced inMod.1 cassette compared to the original one, probably
because the guide strand is correctly cleaved at A or U
at two nucleotides inside from the double strand terminalby this nucleotide modification. Furthermore, AG residues at the two nucleotides downstream of DraIII site
in the original mir-187 sequence were changed to C
residue in Mod.2, 3, and 4. This region is the bindingsite of DGCR8, a partner of Drosha, which is a doublestranded RNA binding protein. The accessibility of
DGCR8 may increase, since the double stranded regionis enlarged by this modification. Indeed, the luciferase
activity was also reduced compared to the original one.
Then, the expression cassette was inserted into the pCAG

RNAi CONSTRUCT BASED ON mir-187

45

Fig. 2. Modification of mir-187 precursor and mir-30 loop sequences for optimizing the knockdown efficacy. A: The modified
nucleotide (s) in original mir-187 sequence was shown in underlined bold character, and siRNA sequence against firefly
luciferase is shown in italic. The loop region of mir-30 was essentially used except for Mod.3. B: The knockdown efficacy
was measures by Dual-Luciferase Reporter Assay System (Promega) by co-transfection of each of RNA polymerase IIdriven miR-187-based siRNA expression construct (5, 50, 150 ng) with target firefly luciferase-expressing pGL3-Control
vector (1.0 µg) and Renilla luciferase-expressing pRL-SV40 vector (10 ng) using human HeLa cells. Luciferase activities
were measured in two to four wells, respectively. Each error bar represents the SD.

expression vector (pCAG-miR-187, Fig. 1B). Subsequently, an EF1-α/EGFP expression cassette was inserted to visualize the efficiency of transfection in vitro.
Furthermore, an SV40/blasticidin gene was inserted forthe establishment of stable cell lines. Consequently, theRNAi vector could be utilized for both in vitro and invivo studies (pGKD-miR-187, Fig.1C). The shRNA sequences against mMc4r or control firefly luciferase (Luc)genes were designed using an algorithm reported previously [22, 29]. The annealed oligonucleotides containing
the miR-30 loop sequence were inserted into the pCAGmiR-187 or pGKD-miR-187 vector in the site createdby a double digest with PshAI and DraIII (pCAG-miRmMC4R, pCAG-miR-Luc, and pGKD-miR-mMC4R).
Knockdown efficacy of pCAG-miR-mMC4R in cultured
cells
We estimated the knockdown efficacy of the pCAGmiR-mMC4R construct against transiently expressed

mMc4r in HEK293 cells. The pcDNA3.1/mMC4R expression construct was transiently co-transfected into
HEK293 cells with an empty pCAG vector without themir-187 cassette, the pCAG-miR-mMC4R construct, or
the pCAG-miR-Luc construct. One day after transfection, qPCR analyses were performed to measure mMc4r
and human β-actin expression. Co-transfection of thepCAG-miR-mMC4R construct reduced the expressionof mMc4r mRNA by 80%, while co-transfection with
pCAG-miR-Luc had no effect (Fig. 3). These results
indicate that the pCAG-miR-187 expression cassette
effectively induced RNAi and specifically repressedtarget gene expression in vitro.
Generation of GKD-miR-mMC4R transgenic mice
We generated four transgenic founder mice carrying
the GKD-miR-mMC4R gene (Suppl. Fig. 1A), and investigated whether the miR-mMC4R construct could
knock down endogenous expression of mMc4r in vivo.

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M. KATO, ET AL.

Fig. 3. Knockdown efficacy of pCAG-miR-mMC4R in HEK293 cells. HEK293 cells wereco-transfected with pcDNA3.1-mMC4R and either pCAG-miR-187 empty vector,
pCAG-miR-mMC4R, or pCAG-miR-Luc. One day after transfection, total RNA wasextracted and qPCR analyses for mouse Mc4r and human GAPDH were performed.
The mean of three independent assays were shown. Each error bar represents the SE.

Of these transgenic mice, weight gain was observed inonly one transgenic male (10L, Suppl. Fig. 1B). Correspondingly, the highest expression among the foundermice of the miR-mMc4r precursor RNA was detected in
the hypothalamus of mouse 10L (Suppl. Fig. 2). Fromthese results, we concluded that the obese phenotype
observed in 10L can be explained by the greater expression of the miR-mMc4r precursor RNA in the hypothalamus (Suppl. Fig. 2). The body weight of the transgenic mouse line (10L) was further compared to thoseof wild-type sibs from 6 to 31 weeks of age. A significant
increase in the body weight of the transgenic micestarted at 11 weeks, and transgenic mice were 30%
heavier than non-transgenic littermates at 31 weeks (Fig.
4).
RNA expression in GKD-miR-mMC4R transgenic mice
Small RNAs were purified from the hypothalamus,
striatum, hippocampus, and pre-frontal cortex regionsof the brains of both the GKD-miR-mMC4R transgenic
mice (10L) and wild-type sibs, and were examined by
Northern blot analysis. Although a faint backgroundsignal was also detected in the brain of wild type mouse,a sense probe detected the strong signals of the guidestrand of mMc4r siRNA in all of the brain regions extracted from the transgenic mice (Fig. 5). In addition,
the mMc4r transcripts in these four brain regions of thetransgenic mice were compared to those in the wild-typesibs using qPCR. A significant reduction in the expres-Fig. 4. Weight gain of GKD-miR-mMC4R transgenic mice. The
symbols indicate the first week that body weight differedsignificantly between transgenic (Tg; n=9) and non-transgenic (nTg; n=15) mice (*; P<0.05, †; P<0.01). Error bars
indicate SE.

sion of mMc4r mRNA (20–30%) was obtained in all fourbrain regions of the transgenic mouse line (Table 1). Thispartial decrement of mMc4r mRNA in the brains of thetransgenic mice strongly indicated that the siRNA produced from the GKD-miR-mMC4R transgene attenuated the expression of mMc4r mRNA in vivo.

47

RNAi CONSTRUCT BASED ON mir-187

Fig. 5.	Small RNA analyses of GKD miR-mMC4R mice. Northern blots for guide strand of mMC4R siRNA and miR-16
in various brain regions of miR-mMC4R transgenic mice. Either 6 µg (prefrontal cortex, striatum, or hippocampus)
or 2 µg (hypothalamus) of the small RNA fraction was separated on 15% polyacrylamide-urea gels. 1: miR-mMC4R
transgenic, 2: wild-type, 3: wild-type + 1 ng synthetic siRNA for mMc4r.

Table 1. qPCR analyses of GKD-miR-mMC4R transgenic mice
Relative expression of mMc4r(mouse Mc4r / mouse β-actin)
Hypothalamus
Prefrontal cortex
Striatum
Hippocampus

non-Tg (n=9)

Tg (n=15)100 ± 2.47
100 ± 4.21
100 ± 3.51
100 ± 3.73

80.6 ± 4.34 **80.2 ± 8.30 *70.1 ± 5.32 **73.9 ± 5.53 **Mean ± SE, *; P<0.05, **; P<0.01 (t-test).

Food consumption and metabolic phenotypes of GKDmiR-mMC4R transgenic mice
Next, we examined the hyperphagic phenotype in malelitters of transgenic mice. The total food consumptionof 37- to 41-week-old transgenic mice was determinedover 9 days. The knockdown of mMc4r resulted in asignificant increase (9%) in normal diet consumption
over that of non-transgenic littermates (Table 2). To further understand the obese phenotype of transgenic mice,
we performed a number of assays of parameters related
to obesity: oral glucose tolerance tests (OGTT); measurements of glucose, triglyceride (TG), insulin, free fatty
acid (FFA), total cholesterol, and leptin in serum; measurement of tissue weight for mesenteric fat, pancreas,
and liver; and measurement of leptin and TG levels in
the latter two tissues, respectively. There were no significant differences in glucose, TG, FFA, or total cholesterol levels between fasting transgenic and non-transgenic mice. However, insulin and leptin weresignificantly elevated in transgenic mice, by 6-fold and2.5-fold, respectively.

In transgenic mice, the tissue weights of pancreas,
liver, and mesenteric fat were significantly increased.
The content of insulin in pancreas showed a 3.5-foldincrease in transgenic mice. Furthermore, liver TG levels were also significantly elevated in transgenic mice
(Table 2). The AUC0~2 h of the glucose response during
OGTT tended to increase in transgenic mice compared
to non-transgenic mice but was not significantly different (Suppl. Fig. 3).
From these observations, we concluded that GKDmiR-mMC4R transgenic mice had a phenotype of hyperinsulinemia, fatty liver, functional disorder of thepancreas, and the accumulation of mesenteric fat.
In conclusion, we have determined that a mir-187based expression construct is practically useful for bothin vitro and in vivo RNAi. The expression cassette introduced into mice could express functional siRNAs against
target genes in tissues. While transgenic miR-mMC4R
mice had the obese phenotype previously seen in knockout mice, this has revealed that such a phenotype may
be the result of as little as a 20–30% partial knockdownof mMc4r expression in vivo.
Discussion
We have described the development of a new siRNAexpressing cassette, and applied it in RNAi in vitro andin vivo. The method depends on the expression of specific siRNAs within mir-187 precursor sequences. Because mir-187 has recognition sites for the restriction
enzymes DraIII and PshAI in its flanking regions, syn-48

M. KATO, ET AL.
Table 2. Food consumption and metabolic phenotypes of GKD-miR-mMC4R transgenic mice
non-Tg (n=9)

Tg (n=15)

35.6 ± 3.4

38.4 ± 3.3

*

Serum
Glucose (mg/dl)
Trigliceride (mg/dl)
Insulin (ng/ml)
Free fatty acid (uEq/l)
Total cholesterol (mg/dl)
Leptin (ng/ml)

85.12 ± 3.20
71.8 ± 1.8
3.2 ± 0.3
1,369.3 ± 44.4
181.9 ± 10.8
19.8 ± 2.9

89.12 ± 3.76
73.6 ± 3.6
20.0 ± 6.7
1,483.4 ± 98.0
199.4 ± 11.6
50.8 ± 15.7

NS
NS
*
NS
NS
*

Pancreas
Insulin (ng/ml/g tissue)
Tissue weight (g)58,387.2 ± 3,246.2
0.372 ± 0.013

203,192.5 ± 28,998.7
0.399 ± 0.016

**
*

Liver
Trigliceride (mg/g tissue)
Tissue weight (g)28.3 ± 1.3
1.387 ± 0.060

38.1 ± 2.0
2.258 ± 0.174****

Messenteric Fat
Tissue weight (g)0.990 ± 0.067

1.653 ± 0.070**

Food consumption
Total food intake for 9 days (g)Mean ± SE, *; P<0.05, **; P<0.01 (t-test).

thetic hairpin DNA sequences can be directly inserted
into the vectors (Fig. 1A). This enables high-throughput
construction of siRNA-expressing libraries.
A previous study showed that the body weights ofhomozygous mMc4r mutant mice were 50% heavier thanthose of wild-type mice at 15 weeks of age, and heterozygous mice showed an intermediate weight gain of
approximately 25% [11]. In the present study, miRmMC4R transgenic mice, whose mRNA level of mMc4r
decreased by 20~30% (Table 1), body weights were 15%
heavier than those of wild-type littermates at 15 weeks
of age (Fig. 4). Furthermore, in the abovementioned
previous study, food consumption of homozygous
mMc4r knockout mice increased by 40% over that ofwild-type mice [11], whereas the rate of increase in miRmMC4R transgenic mice in our study was 9% (Table 2).
We conclude that the degree of mMc4r-downregulationand the phenotypes of obesity and hyperphagia are directly proportional.
Although the miR-mMC4R expression construct mediates effective (around 80%) knockdown in cell culture(Fig. 3), the knockdown efficiency in transgenic mice
was limited to about 20~30% (Table 1). One potential
reason for this discrepancy is that the expression levelof transgenic siRNA against mMc4r was not enough toachieve effective knockdown in vivo. In Northern blot
analysis, approximately 1 ng siRNA was detected in 6
µg small RNA extracted from striatum (Fig. 5). The 6µg small RNA was obtained from about 80 mg striatal
tissue (data not shown). The concentration of 1 ng siRNA/ 80 mg tissue is comparable with ~1 nM siRNA. There
are two main reasons that explain the low-level expression of transgenic siRNA. One possibility is that a large
amount of shRNA expressed from the CAG promoter
could saturate the activity of exportin-5, a rate-limiting
factor required for nuclear export of pre-miRNAs [5].
Such high shRNA expression may inhibit endogenous
miRNA functions required for normal embryogenesis
and development [30]. Consequently, only transgenic
mice with low shRNA expression would survive to birth.
In fact, after microinjection of the CAG-EGFP transgene,bright EGFP expression in embryos can be observed atearly developmental stages, from the 2-cell stage to blastocysts [13]. To overcome this possible problem, conditional expression constructs, with inducible or stagespecific RNA pol II promoters, may be valuable.
Another possible reason for the low-level expression oftransgenic siRNA is that the silencing of transgene by
the vector sequences or by the random and multi-copy
integration [4, 24, 28]. Especially, the vector used in thisstudy (Fig. 1C) contained prokaryote-derived elementsfrom pBluescript, therefore, it’s plausible to remove suchbacterial sequences before injection into mice eggs.
Although the obese phenotypes of miR-mMC4R transgenic mice were similar to those of mMc4r knockout
mice in terms of hyperinsulinemia [1] and fatty liverRNAi CONSTRUCT BASED ON mir-187[27], the serum levels of glucose were normal in miRmMC4R transgenic mice (Table 2). In previous studies,
pre-obese mMc4r homozygous knockout mice did notdisplay hyperglycemia and hyperinsulinemia, although
they were already significantly heavier than heterozygous knockout and wild-type mice [1]. mMc4r knockout
mice develop both hyperglycemia and hyperinsulinemia[11], whereas the miR-mMC4R transgenic mice in ourstudy exhibited only hyperinsulinemia (Table 2). Reexpression of mMc4r specifically in the lateral hypothalamic area (LHA) improves glucose metabolism in obese
mMc4r knockout mice, without affecting body weightor circulating insulin levels [21]. It is possible that a
partial knockdown of mMc4r (20% to 30%) did not affect
LHA signaling in the regulation of glucose homeostasis.
The partial knockdown by the mir-187 expression construct in vivo may be a powerful tool to investigate therelationship between the level of gene expression andthe phenotype of interest.
In conclusion, we developed a new siRNA-expression
construct based on the mir-187 precursor. Transgenic
mice with partial knockdowns offer valuable functional
information. Such mir-187 precursor-based constructs
will provide new tools for validating drug target genes
in vitro and in vivo, and for generating hypomorphic
animal models.
Acknowledgments
We thank Mrs. K. Munakata and Mrs. M. Kamizono
for excellent technical assistance. This work was supported by grants from the Ministry of Education, Culture,
Sports, Science, and Technology of Japan (21310123,
21115004, and 16H14640) and the Cell Innovation Project (MEXT) to K.U.-T, and by a research fund provided
by Mitsubishi Kagaku Institute of Life Science to H. T.
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