A Novel Dual-Color Reporter for Identifying Insulin-
Producing Beta- Cells and Classifying Heterogeneity of
Insulinoma Cell Lines
Nan Sook Lee1*, Joyce G. Rohan1¤a, Madison Zitting2, Sonia Kamath1, Andrew Weitz2, Arnold Sipos3,
Paul M. Salvaterra4, Kouichi Hasegawa5¤b, Martin Pera5¤c, Robert H. Chow1
1Department of Physiology & Biophysics and Zilkha Neurogenetics Institute, University of Southern California, Los Angeles, California, United States of America,
2Department of Biomedical Engineering, University of Southern California, Los Angeles, California, United States of America, 3School of Medicine, University of Southern
California, Los Angeles, California, United States of America, 4Division of Neuroscience, Beckman research Institute of the City of Hope, Duarte, California, United States of
America, 5Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Research, University of Southern California, Los Angeles, California, United States of
Many research studies use immortalized cell lines as surrogates for primary beta- cells. We describe the production and use
of a novel ‘‘indirect’’ dual-fluorescent reporter system that leads to mutually exclusive expression of EGFP in insulin-
producing (INS+) beta-cells or mCherry in non-beta-cells. Our system uses the human insulin promoter to initiate a Cre-
mediated shift in reporter color within a single transgene construct and is useful for FACS selection of cells from single
cultures for further analysis. Application of our reporter to presumably clonal HIT-T15 insulinoma cells, as well as other
presumably clonal lines, indicates that these cultures are in fact heterogeneous with respect to INS+phenotype. Our
strategy could be easily applied to other cell- or tissue-specific promoters. We anticipate its utility for FACS purification of
INS+and glucose-responsive beta-like-cells from primary human islet cell isolates or in vitro differentiated pluripotent stem
Citation: Lee NS, Rohan JG, Zitting M, Kamath S, Weitz A, et al. (2012) A Novel Dual-Color Reporter for Identifying Insulin-Producing Beta- Cells and Classifying
Heterogeneity of Insulinoma Cell Lines. PLoS ONE 7(4): e35521. doi:10.1371/journal.pone.0035521
Editor: Francis C. Lynn, University of British Columbia, Canada
Received December 6, 2011; Accepted March 17, 2012; Published April 18, 2012
Copyright: ? 2012 Lee et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Wright Foundation (USC). The funders had no role in study design, data collection and analysis, decision to publish, or
preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: email@example.com
¤a Current address: Department of Neuroscience, Cell Biology and Physiology, Wright State University, Dayton, Ohio, United States of America
¤b Current address: Institute for Integrated Cell-Material Science, Kyoto University, Kyoto, Japan
¤c Current address: The Stem Cell Program, University of Melbourne, Melbourne, Australia
Diabetes prevalence is increasing dramatically worldwide and
severe co-morbidities persist, despite the availability of insulin
treatment. Cell replacement strategies are thus being developed to
treat this metabolic disease. A key treatment step will be
production of sufficient quantities of fully functional pancreatic
beta- or beta-like-cells suitable for replacing missing or defective
beta-cells. This goal has stimulated renewed interest in under-
standing human islet cell biology. However, because of the
difficulty and high cost associated with isolating human islets, most
studies focus on in vitro characterization of immortalized human or
animal cell lines as surrogates for primary beta-cells.
Rodent insulinoma cell lines derived from cancers arising after
radiation treatment (rat RIN, INS-1, CRI-G1) or viral transfor-
mation (hamster HIT, bHC) have been especially useful models
of beta-cell biology; at the time of their establishment, they
exhibit high levels of insulin production and glucose respon-
siveness [1,2,3,4]. However, both of these pancreatic beta-cell
attributes are lost with time in culture and increased numbers of
cell passages . Unfortunately, the generation and characteriza-
tion of human insulinoma or beta-cell-derived cell lines that
preserve normal glucose responsiveness has not been reported.
The hamster insulinoma cell line, HIT-T15, has been one of the
most extensively studied beta-cell-like models. HIT-T15 cells
exhibit glucose-stimulated insulin secretion and contain mem-
brane-bound secretory granules , similar to those seen in
normal islet beta-cells. HIT-T15 cells were originally produced
by SV40 transformation of pancreatic beta-cells, followed by
serial selection of clonal lines expressing the glucose-responsive
In this study, we describe the development and application of a
new dual-color fluorescent reporter system for identifying insulin-
producing (INS+) beta- and non-insulin-producing (INS-) cells.
Our reporter contains a single transgene with two expression
cassettes. The first is a fragment of the human insulin gene
promoter (phINS) that drives expression of Cre recombinase
protein exclusively in INS+cells. The second contains the CMV
promoter and an mCherry coding region flanked with LoxP (L)
sites, followed by an EGFP coding region. In cells with active
insulin promoter activity, the Cre protein excises the mCherry
coding region, and the cells exhibit green fluorescence. In cells
PLoS ONE | www.plosone.org1April 2012 | Volume 7 | Issue 4 | e35521
with no insulin promoter activity, the mCherry coding region is
not excised, so the cells exhibit red fluorescence. This new
‘‘indirect’’ reporter strategy uses mutually exclusive expression of
green or red fluorescence to eliminate ambiguity observed when a
human insulin promoter directly drives expression of EGFP in
combination with a CMV-regulated mCherry. Distinguishing
INS+from INS-cells with the ‘‘direct’’ strategy depends on
identifying cells that are doubly fluorescent and often leads to
ambiguous results—the relative levels of fluorescence for the two
reporter colors can be highly variable (due to variability in the
relative strength of the two promoters driving fluorescent protein
expression, differences in the relative fluorescence intensities, and/
or relative rate of degradation of the proteins). Our ‘‘indirect’’
dual-color system, in contrast, reports all cells that have been
transduced or transfected, so efficiency of transduction/transfec-
tion is easily calculated.
Regardless of which fluorescent protein is expressed, expression
is under control of the same CMV promoter. We thus observe
only a single color for each phenotype that reports successful Ins
gene expression by a discrete change in color from red to green. In
addition, our approach results in separate colors arising from the
same transfected cells using a single transgene construct, which is
not possible with a single-color reporter.
We prepared HIT-T15 cells stably transfected with our new
reporter construct. Contrary to expectations, we observed that
these presumably clonal cells are in fact heterogeneous with
respect to a variety of beta-cell-like biochemical and electrophys-
iological phenotypes. After FACS isolation of green fluorescent
cells, we obtained a stable, homogeneous population of insulin-
producing cells with beta-like phenotypes. Since our reporter
system uses the human insulin promoter, we anticipate its utility to
identify and FACS purify INS+and glucose-responsive beta-like-
cells from primary human islet cell isolates or in vitro differentiated
human pluripotent stem cells.
Specificity of the proximal 378-nt region in the human
The complete human insulin promoter/enhancer is estimated
to be ,4 kb in length [8,9]. The proximal 378-nt region (2363 to
+15) is highly conserved between humans and rodents [10,11]. It
contains important regulatory motifs (Fig. 1a) previously used to
make reporters for identifying INS+cells [6,12]. We first
confirmed the utility of this region to report insulin production
by transfecting a phINS-EGFP construct (Fig. 1b, ) into the
HIT-T15 hamster insulinoma clonal cell line (insulin-producing)
, as well as the NT-2 human embryonic carcinoma cell line
(non-insulin-producing). As expected, only HIT-T15 cells exhib-
ited green fluorescence (Fig. 1c). In contrast, using the ubiquitous
CMV promoter instead of the insulin promoter to drive EGFP
expression resulted in fluorescence of both cell types (Fig. 1c). We
therefore concluded that this 378-nt promoter region is sufficient
for marking insulin-producing cells, and we used this region to
construct additional reporters.
‘‘Direct ’’ dual-color reporter system
We modified phINS-EGFP by introducing a dsRed reporter
cassette under control of the CMV promoter, reasoning that this
could be used to control for transfection efficiency and to identify
non-insulin producing cells. We expected INS2cells to exhibit
only red fluorescence, and INS+cells to exhibit both red and green
(yellow) fluorescence. Transient (data not shown) and stable
transfection of phINS-EGFP-CMV-dsRed (Fig. 1d) into HIT-T15
cells resulted in cells that displayed red or yellow fluorescence
(Fig. 1e). However, a small population of cells unexpectedly
appeared to express primarily green fluorescence (Fig. 1e). We
reasoned that this unexpected fluorescence was caused by unequal
accumulation of fluorescent reporter protein in certain cells with
Figure 1. Specificity of the human insulin promoter (378-nt region) to identify insulin-producing cells and a direct dual-color
reporter system. (a) Schematic picture of the proximal 378-nt region (2363 to +15) of the human insulin promoter containing several regulatory
motifs for expression of the insulin gene. (b) The phINS-EGFP construct  used to express EGFP in insulin-producing cells. (c) phINS-EGFP was
transfected into HIT-T15 or NT-2 cells. After 2 days, some HIT-T15 cells expressed EGFP, but NT-2 cells did not. When pCMV-EGFP was transfected,
both cell lines expressed EGFP, suggesting the specificity of the 378-nt region to identify insulin-producing cells. (d) Schematic picture of a direct
dual-color reporter that contains a human insulin promoter driving expression of EGFP and a CMV promoter driving expression of dsRed (phINS-
EGFP-CMV-dsRed). (e) Stable transfection of phINS-EGFP-CMV-dsRed into HIT-T15. Many cells displayed red or yellow fluorescence, but a small
population of cells unexpectedly appeared to express primarily green fluorescence (white arrows).
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different levels of expression  or unequal catabolism of the two
fluorescent proteins . Alternatively, transcriptional inactivation
of promoters could have occurred during selection of stably
transfected cells [15,16,17,18]. Nevertheless, the presence of the
green fluorescent cells complicated clear identification of cells with
INS+versus INS2phenotypes, so we decided to try a new
‘‘Indirect’’ dual-color reporter system
Rather than use the insulin promoter to directly drive EGFP
expression, we tested an alternate strategy in which the insulin
promoter drives expression of Cre recombinase, leading to
excision of an mCherry coding region located between CMV
promoter and EGFP coding region. The mCherry coding region is
followed by a stop codon; thus, the new design leads to mutually
exclusive expression of either red or green fluorescent proteins.
To confirm that insulin promoter activity led to expression of
Cre recombinase, we constructed a plasmid containing the Cre
gene under the human insulin promoter (phINS-Cre-pA; Fig. 2a).
This plasmid was transiently transfected into HIT-T15 cells, as
well as MDA-MB-231 cells (non-insulin-producing negative
controls). Roughly 70% of HIT-T15 cells became Cre+(red),
while MB-231 cells did not show Cre expression (Fig. 2b). When
transiently transfected with pEGFP-N1, ,70% of cells showed
EGFP expression for both cell lines, indicating similar transfection
efficiencies (Fig. S1). When HIT-T15 cells were transiently co-
transfected with phINS-Cre and pEGFP-N1 (Fig. 2c), ,70% of
HIT-T15 cells were Cre+(red), but not all red cells were green.
This suggests that not all cells were transfected with both plasmids
and motivated us to use a single construct containing a
combination of both cassettes.
To make the single construct, we first constructed a plasmid
containing only the fluorescent reporters (pCMV-L-mCherry-L-
EGFP) (Fig. 3a). This plasmid was transiently transfected into
human fibroblasts (a non-insulin-producing negative control), as
well as in HIT-T15 cells and the rat insulinoma cell line, INS-1
823/13 . The latter two cell lines are INS+but should not
show any red-to-green color change in the absence of a source of
Cre. As expected, all transfected cells showed only red fluorescence
We next constructed [pA-Cre-phINS(reverse orientation)]-
pCMV-L-mCherry-L-EGFP by inserting a phINS-Cre-pA cas-
sette fragment (Fig. 2a) upstream of pCMV-L-mCherry-L-EGFP
in reverse orientation (Fig. 3b). Transfected INS+cells should
activate Cre recombinase expression and excise the mCherry
reporter by Cre-LoxP recombination, thus converting red
fluorescence to green. In contrast, transfected INS–cells should
only exhibit red fluorescence. We refer to this reporter as
‘‘indirect,’’ since phINS does not directly drive EGFP expression,
but rather changes the color of the reporter being expressed under
control of the ubiquitous CMV promoter.
Transiently transfected fibroblasts showed only red fluorescence
(Fig. 3c), indicating the absence of any Cre-mediated recombina-
tion. In contrast, transiently transfected HIT-T15 and INS-1 823/
13 cells showed either red or green fluorescence, indicating Cre-
mediated excision of the mCherry cassette in a subset of the
transfected cells. We also observed a small number of HIT-T15
and INS-1 823/13 cells with yellow fluorescence (Fig. 3c),
indicating expression of both red and green fluorescent proteins
(Fig. 3c). We imaged the cells with confocal microscopy to confirm
that the yellow cells showed both green and red fluorescence,
rather than being two separate cells stacked atop one another
(Fig. 3d, Fig. S2). The yellow fluorescence may be a technical
artifact due to unequal copy numbers of our test plasmid in
different cells  or unequal intracellular catabolism of red versus
green fluorescent proteins . We therefore produced stably
transfected HIT-T15 cells using G418 selection for a neomycin
resistance gene (Neo) incorporated in our plasmid to present 1–2
copies of the plasmid.
We expected all stably transfected cells to exhibit green
fluorescence, since HIT-T15 is believed to be a clonal model of
beta-like-cells and all should have activated the insulin promoter
and expressed Cre . However, we found that only ,70% of
the stably transfected cells were green, indicating mCherry
excision (Fig. 3e, left panel). Another ,10% were red, indicating
the absence of mCherry excision and suggesting that the insulin
promoter was never activated. The remaining ,20% of cells
exhibited no fluorescence, suggesting that transcriptional inacti-
vation of the CMV promoter might have occurred during
Figure 2. Expression of Cre recombinase from phINS-Cre
plasmid. (a) Schematic picture of phINS-Cre construct. (b) Immuno-
fluorescence analysis of HIT-T15 and MB-231 cells transiently transfect-
ed with phINS-Cre. The red color indicates Cre recombinase expression.
Scale bar: 10 mm. (c) Immunostaining of HIT-T15 cells transiently co-
transfected with phINS-Cre and pEGFP-N1. The red color indicates Cre
recombinase expression (Cy-3), and the green is a marker for co-
transfection. (d) Immunostaining of stable HIT-T15 FACS-sorted GFP+
and mCherry+cells with and without the primary anti-Cre antibody. The
green colors in the top and middle panels were visualized as EGFP
expression, and the red (Cy-3) as Cre recombinase expression. The
stable FACS-sorted mCherry+cells in the bottom panels did not express
Cre recombinase (no green, Cy-2).
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selection of stably transfected cells, although conflicting data about
the activity of the CMV promoter in rodent undifferentiated
[15,16,17,18]. No yellow fluorescent cells were observed (Fig. 3e).
These results suggest that at least two phenotypically heteroge-
neous cells arise in the HIT-T15 cell culture model. We could not
select stably transfected INS-1 823/13 cells because they already
contain a pCMV8/INS/IRES/Neo insertion .
FACS sorting of the stably transfected HIT-T15 cells followed
by q-RT-PCR analysis of each population directly confirmed their
heterogeneity with respect to INS gene expression (Fig. 4). High
levels of INS transcript were detected in the green population
(Fig. 4b), while larger amounts of glucagon (GCG) were detected
in the red population (Fig. 4c). Pdx-1 transcripts were expressed at
higher levels in GFP+cells relative to mCherry+cells (Fig. 4d). The
trends were similar, regardless of the reference gene (GAPDH vs.
Cyclophilin). FACS-sorted stable GFP+cells showed much weaker
Cre recombinase (red, Cy-3) expression (Fig. 2d) (1–2 copies of the
plasmid) relative to the transiently transfected cells (several copies)
(Fig. 2b & c), confirming INS+phenotype. In contrast, FACS-
sorted mCherry+cells did not express Cre recombinase (no green,
Cy-2) (Fig. 2d, bottom panel), indicating INS2phenotype.
Furthermore, FACS-sorted GFP+and mCherry+cells grown
separately in culture maintained their fluorescence throughout five
subsequent passages, thus appearing to be stable. These data
establish that the color shift from red to green using our reporter
system faithfully indicates the status of INS gene expression. They
also suggest that the original HIT-T15 population may have been
heterogeneous with respect to INS+phenotype (i.e., a population
of GCG+cells was also present), or perhaps cells change from
INS+to GCG+in culture by overexpression of aristaless-related
homeobox (Arx) (i.e., converting beta-cells into alpha-cells
[21,22]). Figure 4e supported the latter case, but we are further
characterizing both cell populations by RNA sequencing analysis.
Electrophysiological characteristics of FACS-sorted GFP+
We further characterized the FACS-sorted INS-expressing
green and non-INS-expressing red cells electrophysiologically.
Using a standard whole-cell patch- clamp configuration, we found
that the green cells exhibited more than a two-fold greater inward
current than the red cells (Fig. 5 a–c; P,0.0001). The kinetics of
the inward current were characteristic of Ca2+channels, an idea
further supported by the observation that addition of Cd2+to the
bath effectively blocked the current (Fig. 5d). We also found that a
greater proportion of stimulated green cells underwent exocytosis
Figure 3. ‘‘Indirect’’ dual-color design of the reporter construct. (a) Transfection of cells with CMV-L-mCherry-L-EGFP. Without Cre
recombinase in the construct, all cells exhibited red fluorescence. (b) Schematic and anticipated behavior of pA-Cre-phIns-CMV-L-mCherry-L-EGFP.
INS+beta-cells transfected by this plasmid should activate Cre recombinase expression and excise the mCherry reporter by Cre-LoxP recombination,
thus converting red fluorescence to green. In contrast, transfected INS2non-beta-cells should only exhibit red fluorescence. (c) After transfection of
cells with pA-Cre-phIns-CMV-L-mCherry-L-EGFP, HIT-T15 and INS-1 expressed EGFP, indicating that mCherry was excised by Cre recombinase
expressed under the human insulin promoter. This demonstrates the reporter’s specificity to produce EGFP in only INS+cells. (d) Highly magnified
(636) projection images of two yellow cells marked as the white square with a white arrow in Fig. 2c from Z-stacks (106), illustrating both red and
green fluorescence. In the middle panel, the 3D picture shows co-localization of both colors (B, bottom; T, top). (e) Stable HIT-T15 cell population
containing the ‘‘indirect’’ dual-color construct (Neor). ,70% of the stably transfected cells were green, indicating mCherry excision. Another ,10%
were red, indicating the absence of mCherry excision and suggesting that the insulin promoter was never activated. The remaining ,20% of cells
exhibited no fluorescence, suggesting transcriptional inactivation of the CMV promoter. The right panel depicts the cells after more than 6 days in
culture, compared to cells at 1 day after splitting shown in the left panel. No yellow fluorescent cells were observed. These results suggest that at
least two phenotypically heterogeneous cells arise in the HIT-T15 cell culture model.
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compared to red cells [74% (21 out of 30) versus 28% (7 out of
25)], as inferred from changes in membrane capacitance following
trains of eight depolarizing pulses (from 280 to 0 mV, 100 ms
duration; Fig. 5e & f). Adding Cd2+blocked changes in membrane
capacitance, as would be expected for insulin secretion, which is
known to be predominantly Ca2+-dependent (Fig. 5d).
The main voltage-gated Ca2+channels controlling insulin
secretion in beta-cells are L-type channels that can be blocked
by nitrendipine . Addition of 20 mM nitrendipine only
partially blocked the Ca2+current (,50%) but almost totally
eliminated the capacitance increase in green cells (Fig. 5f). This
suggests that more than one type of Ca2+channel is present, but
that L-type Ca2+channels gate the Ca2+entry that is coupled to
exocytosis. There was a greater variability in the amplitude of
calcium currents and capacitance changes in red cells compared to
One characteristic of beta-cells is that intracellular Ca2+levels
([Ca2+]i) rise in response to increased extracellular glucose [24,25].
We used ratiometric imaging of the membrane-permeant
calcium indicator dye, Fura2-AM (Fura2 acetoxymethyl ester
), in cells exposed to either basal (3 mM) or stimulatory
(10–14 mM) levels of glucose. The fluorescence ratio (350 nm/
Figure 4. INS and GCG transcript expression in FACS-sorted GFP+and mCherry+cells using q-RT-PCR. (a) After FACS analysis in stable
HIT-T15 cell population to select GFP+or mCherry+cells, q-RT-PCRs were performed in GFP+and mCherry+cells. (b, c) q-RT-PCR data confirmed that
the green and red cells were insulin- and glucagon-producing, respectively. (d, e) The green and red cells were analyzed for expression levels of Pdx1
(d) and Arx (e) genes by q-RT-PCR. The expression levels of INS, GCG, Pdx1, and Arx transcripts were normalized to the housekeeping genes, GAPDH
and/or Cyclophilin (‘‘MNE’’). The error bars represent standard error (SEM) of the mean (N=3 for each experiment).
Figure 5. Electrophysiological differences between GFP+and mCherry+cells. (a) GFP+cells exhibited significantly greater calcium currents
compared to mCherry+cells (Mann Whitney, P,0.0001, n=19–22). (b) Example current traces from GFP+cells (top panel) and mCherry+cells (bottom
panel). Scale bar: 100 pA, 10 ms. (c) Average current-voltage plot showing greater calcium influx in GFP+cells (closed circles) compared to mCherry+
cells (open circles). (d) Cadmium (Cd2+, 30 mM) blocked inward current in a GFP+cell. (e) Depolarizing trains of 8 pulses (100 ms, 0 mV) induced an
increase in capacitance (corresponding to an increase in exocytosis) from most of GFP+cells (21 out of 30). Only 7 out of 25 cells mCherry+cells
underwent an increase in capacitance when stimulated by 8-pulse depolarizing trains. (f) Capacitance recording from the same GFP+cells with and
without 20 mM Nitrendipine.
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380 nm) increased when a green cell was exposed to the
stimulatory level of glucose (compare the green line in the left
and right panels of Fig. 6a), indicating an increase in [Ca2+]i. The
other colored lines in Figure 6a represent non-fluorescent cells,
which do not exhibit a fluorometric ratio change under the same
conditions. Ratiometric analysis of red cells did not show any
change when shifted from the basal to the stimulatory glucose
concentration (Fig. 6b). We also performed ratiometric imaging on
INS-1 823/13 cells and observed increased [Ca2+]iin both basal
and stimulating glucose concentrations (Fig. 6c). INS-1 823/13
cells, which retain insulin over-expression under a ubiquitous
promoter (in contrast to HIT-T15 cells), thus appear to have
spontaneous [Ca2+]ioscillations that are independent of glucose
Figure 6. Intracellular Ca2+concentration, [Ca2+]ichanges associated with glucose stimulation in different fluorescent cells. (a)
Glucose responsiveness of GFP+stable HIT-T15 cells. The green line represents a GFP+cell in basal (3 mM, left panel) and stimulatory (14 mM, right
panel) levels of glucose. The other lines (red, blue, and orange) represent non-fluorescent cells. (b) No glucose responsiveness of mCherry+stable HIT-
T15 cells was observed. (c) Fluorescence traces from INS-1 cells. Each color represents a different cell, indicating spontaneous Ca2+oscillations arising
from action potentials.
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Table 1 presents a summary of our phenotypic analysis of
FACS-sorted stably transfected HIT-T15 cells. The results support
our conclusion that our new ‘‘indirect’’ dual color reporter is an
effective tool to distinguish a range of beta-cell-like phenotypes.
In the present study, we describe the design and use of a new
‘‘indirect’’ fluorescent reporter system that drives mutually
exclusive expression of either EGFP for INS+cells or mCherry
for INS2cells. This new approach relies on a human insulin
promoter-driven Cre-mediated shift in reporter color from red to
green in single transgene construct. This results in several
advantages over other approaches that use single- or dual-color
reporters to distinguish cellular phenotypes.
A single-color reporter system with the insulin promoter driving
expression of a fluorescent protein [27,28,29] serves to identify a
subset of cells fulfilling two conditions: (1) successful transduction/
transfection and (2) activation of the insulin promoter. However,
this system does not allow for straightforward calculation of the
percentage of transfected/transduced cells. Among those cells that
are successfully transfected/transduced, it is not clear what
fractions have activated the insulin promoter.
The ‘‘direct’’ dual-color reporter systems that have been
described use the ubiquitous CMV or a cell-/tissue-specific
promoter to drive one color and another cell-/tissue-specific
reporter for the second color [30,31,32,33,34,35]. Thus, the cells
in which the cell-/tissue-specific promoter is activated should
express two colors, while all other cells should express only one
color. In principle, this system has the advantage compared to the
single-color system of identifying all cells that have been
successfully transfected or transduced, so the efficiency of
transfection/transduction can be easily calculated. However, a
problem with this system became evident when we employed this
approach (Fig. 1d & e). For cells in which the tissue-specific
promoter had been activated, the relative levels of fluorescence for
the two reporter colors was highly variable, due to variability in
the relative strength of the two promoters driving fluorescent
protein expression, differences in the relative fluorescence
intensities, and/or relative degradation rate of the proteins. In a
small subset of cells, the only fluorescence seen was that expected
to be due to the activation of the cell-/tissue-specific promoter. It is
also possible that in these cells, the CMV promoter was inactivated
The ‘‘indirect’’ dual-color reporter system that we have
introduced reports all cells that have been transduced/transfected
(so efficiency of transduction/transfection is easily calculated), and
it also leads to mutually exclusive marking of cells with one or the
other fluorescent protein. Regardless of which fluorescent protein
gets expressed, expression is under control of the same strong and
ubiquitous CMV promoter. We thus observe only a single color
for each phenotype that reports successful INS gene expression by
a discrete change in color from red to green (Fig. 3e and Fig. 4b).
Another advantage of our indirect dual-color reporter relates to
possible off-target effects of the introduced plasmid or viral vector.
Introduction of plasmid or viral vectors into cells may alter cellular
phenotype [36,37], due to heterologous expression of non-native
viral and inserted proteins. Controlling for such changes using a
single-color reporter is not possible, since different cultures with
control vectors are used to assess phenotypes . In contrast, our
approach results in separate colors arising from the same
transfected cells using a single transgene construct. The color
resulting from the Cre-mediated recombination event (i.e.,
removal of the mCherry cassette) thus allows GFP+cells to serve
as an appropriate control for non-insulin-positive cell phenotypes.
A very similar dual-color system to ours was reported , but the
investigators used two separate lentiviral constructs that could not
eliminate the complexity of cell-to-cell transduction variability
(, Fig. 2c). Nevertheless, our reporter may need to discern
gradients of insulin promoter activity (red-to-green cells over time)
in transiently transfected cells using real-time imaging.
Using our new reporter, we found that presumably clonal HIT-
T15 cells [7,20] were heterogeneous with respect to INS gene
expression (Fig. 3e). Approximately 70% of transfected cells were
INS+beta-like-cells, while roughly 30% were INS2, non-beta-like-
cells (possibly of mixed cell phenotypes). This result might be due
to unanticipated differentiation of the cells into different
phenotypic classes during culture or could possibly indicate a
non-clonal nature of the reference HIT-T15 cell line we obtained
from ATCC. It was recently reported that pancreatic beta-cell
identity is maintained by DNA methylation-mediated repression of
Arx [21,22]. Deletion of DNA methyl-transferase gene in INS+
beta-cells converts them into GCG+alpha-cells by derepression of
Arx transcription repressor in beta-cells [39,40]. The red
population of cells showed a low level of INS and Pdx1 transcript
expression and a high level of GCG and Arx transcript expression
(Fig. 4), suggesting conversion of beta-cells into alpha-cells, likely
by over-expression of Arx through lack of DNA methylation-
mediated repression. Regardless, our results argue that care must
be taken in interpretation of previous publications on gene
expression and functional profiling of HIT-T15 cells [41,42,43]
with respect to identification of genes and properties as being
exclusive correlates of authentic beta-like phenotypes. Some of the
previously reported findings may instead be confounded by the
significant population of non-beta-cells.
Our fluorescent reporter enables rapid identification and FACS
purification of a small percentage of unambiguously identified
INS+beta-like-cells in a mixed population with a significant
proportion of non-beta-like-cells (Fig. 4b). We found that GFP+
cells expressed insulin transcript, while mCherry+cells expressed
glucagon (Fig. 4b & c). Furthermore, in comparison to mCherry+
cells, GFP+cells produced significantly larger inward voltage-gated
calcium currents, glucose-stimulated elevation of [Ca2+]i, and
larger voltage-stimulated secretion (Fig. 5 and Fig. 6). These
characteristics are all indicative of authentic beta-cells [23,44]. In
contrast, mCherry+cells exhibited greater variability in the
amplitude of calcium currents and exocytotic responses, perhaps
indicating a mixture of different cellular phenotypes in this
Table 1. Summary of beta-cell-like phenotypes in FACS-
sorted HIT-T15 cells.
Extent of exocytosisb
increase in [Ca2+]i
Insulin gene transcriptionYes (high level)Yes (low level)
Glucagon gene transcriptionYes (low level)Yes (High level)
aCa2+current density is measured in pA/pF (current divided by capacitance), a
measure of the flow of calcium in the membrane area. GFP+cells showed
12.661 pA/pF, while mCherry+cells showed 560.61 pA/pF (p,0.0001).
bThe percentage of cells exhibiting capacitance increase was 74% for GFP+cells
and 28% for mCherry+cells.
A Dual-Color Reporter for Insulin+Beta Cells
PLoS ONE | www.plosone.org8April 2012 | Volume 7 | Issue 4 | e35521
population. In the future, we will use our reporter to isolate purely
homogeneous beta- or beta-like-cells, which will later be used for
more accurate genetic, epigenetic, and functional profiling. Our
approach should also allow us to compare gene expression and
functional phenotypes of beta- or beta-like-cells with other types of
pancreatic cells that may be present in the mCherry+class of cells
(a, d, and PP cells, or perhaps even exocrine acinar cells).
Human islet transplantation using the Edmonton protocol is an
effective treatment for type I diabetes . Its widespread
applicability, however, is limited because of a scarcity of donor
tissue. Properly and correctly differentiated beta-cells from human
pluripotent stem cells could potentially overcome this limitation.
Most current human pluripotent stem cell differentiation protocols
[45,46] have limited reproducibility, low yield of beta-like-cells,
and most importantly, the absence of glucose responsiveness .
We expect that our reporter will facilitate easy and quick
evaluation of new differentiation protocols designed to produce
clinically useful beta-cells—it could be stably introduced into
human pluripotent stem cells  or transduced into different
stage cells using an adenoviral vector [48,49]. In addition, the
reporter system may be used as a high-throughput screening for
reprogramming non-insulin-producing cells to insulin-producing
cells. Further additions and modifications to our approach could
be easily incorporated, depending on the particular phenotypic
property desired. For example, other promoters could be used to
mark different cell- or tissue-specific lineages and to further test
Materials and Methods
Cell culture, transfection, FACS sorting and q-RT-PCR
HIT-T15  (ATCC, USA), INS-1 823/13 (a gift from Chris
Newgard ) and NT-2 cells  were cultured as descried
previously. Human dermal fibroblasts (ScienCell) were cultured in
MEM alpha medium (Invitrogen) with 0.1 mM MEM nonessen-
tial amino acid, 10% FBS, and penicillin/streptomycin. MDA-
MB-231 cells were cultured in DMEM medium with 10% FBS
and penicillin/streptomycin. All cells were incubated at 37uC with
5% CO2 in a humidified incubator. For all transfection
experiments, unless otherwise specified, we used Lipofectamine
2000 (Invitrogen) using the manufacturer’s instructions. For
making stable HIT-T15 cells, cells were transiently transfected
with our reporter (pA-Cre-pINS-pCMV-loxP-mCherry-pA-loxP-
EGFP-pA). Two days after transfection, cells were split at a 1:10
ratio with fresh growth medium into 10 cm tissue culture dishes
and selected with 800 mg/ml G418 until colonies were visible. The
whole populations of stable transfectants or individual colonies
were screened for expression of fluorescent proteins.
Expression of fluorescent proteins was examined with an
Olympus IX70 fluorescence microscope (Olympus, Japan) or a
Leica TCS SP5 confocal microscope (Leica Microsystems,
Germany). Images were analyzed and edited in MetaMorph
For cell sorting, cells were trypsinized after imaging, suspended
in medium, and subjected to FACS sorting for GFP+and
mCherry+cells. FACS was performed by the USC FACS Core
Facility, using a BD FACSAria cell sorter. After cell sorting, total
RNAs were isolated from GFP+or mCherry+cells using Trizol
(Invitrogen). After digestion of total RNAs with Turbo DNase
(Ambion), cDNAs were made from the total RNAs with an iScript
cDNA synthesis kit (Bio-Rad), according to the manufacturer’s
instructions. The cDNA was diluted three-fold with water prior to
quantitative PCR (q-PCR) analysis. Gene-specific q-PCR primer/
probe sets (customized TaqMan Gene Expression Assays, Applied
Biosystems) for hamster INS, GCG, GAPDH, Cyclophillin, and
human Pdx-1 and equivalent amounts of cDNA generated as a
template were used for q-PCR. Reactions were performed in for
each sample using TaqMan Universal PCR Master Mix with a
CFX-96 system (Bio-Rad). The q-PCR was performed using the
manufacturer’s instructions. For each sample, expression of
marker genes was normalized to GAPDH or Cyclophillin. Data
are expressed as a relative expression level. For detection of Arx
that is not well studied in the hamster system, we performed q-
TACCC; R: 59-TCTGTCAGGTCCAGCCTCAT ) as de-
scried previously .
Immonofluorescent staining and confocal microscopy
Cells grown on sterilized, 0.4% gelatin-coated patch-clamp
dishes were washed with PBS and fixed with 4% paraformalde-
hyde for 20 minutes at room temperature. For detection of Cre
nuclear proteins, dishes were permeabilized for 10 minutes with
0.25% Nonidet P-40 (NP40). Cells were blocked overnight at 4uC
with 5% fetal goat serum, 1% bovine serum albumin, and 0.1%
Triton X-100, and incubated for 1 hour with the primary mouse
monoclonal anti-Cre antibody (Sigma-Aldrich, a gift from Le Ma)
diluted in blocking solution (1:500). Cells were then washed and
incubated for 1 hour with the secondary anti-mouse Cy-2 or Cy-3
conjugated antibody (1:250; Jackson ImmunoResearch, a gift from
Le Ma). Images were taken using a Leica TCS SP5 confocal
microscope. EGFP and mCherry were visualized by endogenous
Fluorescent signal of EGFP and mCherry in yellow cells were
visualized by a Leica TCS SP2 AOBS confocal microscope system
(Leica-Microsystems, Heidelberg, Germany) using 106 and 636
magnifications. A Leica DM IRE2 inverted microscope was
powered by Argon and HeNe lasers for the detection of EGFP
(excitation at 488 nm, emission at 495–550 nm) and mCherry
(excitation at 594 nm, emission at 600–750 nm) fluorescence.
Images were collected in xyz series and analyzed by Leica LCS
imaging software (LCS 3D, Process, and Quantify packages).
DNA constructs and expression
All constructs were made using standard molecular cloning
methods. To construct phINS-EGFP-pA-pCMV-DsRed2-pA,
plasmid phINS-EGFP  was digested using SalI and blunted at
both sites with Klenow DNA polymerase (NEB), obtaining phINS-
EGFP-pA fragment. The fragment was then subcloned upstream
of CMV promoter in pDsRed2-N1 (Clontech) digested using AseI
and blunted at both sites with Klenow.
For constructing pCMV-L-mCherry-pA-L-EGFP-pA, a frag-
ment of L-mCherry-pA-L containing HindIII and XhoI restriction
enzyme sites at each 59 and 39 ends, respectively, was obtained by
polymerase chain reaction (PCR) using a template (lox2272-
mCherry-loxP in pBlue-script SK, a gift from Li Zhang ), and
CACCATGGTGAGCAAGGG- CGAGGA) and reverse (59-
TGTCGAGGCCGCGAATTAAAAAACC) primers. Underlined
sequences are the LoxP site and its reverse complement, and the
bold capitalized ATG is the start codon of mCherry. The bold
fonts are the restriction enzyme sites for digestion. The PCR
product was cut by HindIII plus XhoI and blunted at the XhoI site
with Klenow. The HindIII-XhoI (blunt) fragment was inserted into
pEGFP-N1 (Clontech) digested with HindIII plus SalI and blunted
at SalI site with Klenow.
To obtain plasmid phINS-Cre-pA, a Cre-pA fragment was
generated by PCR using pCMV-Cre-pA (a gift from Li Zhang) as
A Dual-Color Reporter for Insulin+Beta Cells
PLoS ONE | www.plosone.org9April 2012 | Volume 7 | Issue 4 | e35521
a template, and forward: 59-ggaattc- ccgcgg ATGCCCAAGAA-
GAAGAGGAAGGTGTC, reverse (59-gaattccttaaggagctc GAA-
CAAACGACCCAACACCCGTG) primers. After digesting with
SacII and AflII, the PCR product was inserted downstream of
human INS promoter in phINS-EGFP that was digested with SacII
and AflII and removed EGFP-pA by gel extraction, generating
plasmid phINS-Cre-pA. To combine phINS-Cre-pA with pCMV-
L-mCherry-pA-L-EGFP-pA, phINS-Cre-pA fragment was ob-
tained from plasmid phINS-Cre-pA by digesting with SalI plus
AflII and blunted at both sites with Klenow. The fragment was
inserted in the reverse orientation (overwhelmingly, but not in the
right orientation) upstream of CMV promoter in pCMV-L-
mCherry-pA-L-EGFP-pA digested with AseI and blunted at both
sites with Klenow. DNA sequences were confirmed using standard
sequencing protocols (USC DNA Sequencing Core Facility).
Electrophysiology of HIT T15 cells
Cells were patch-clamped 2–4 days after plating. Current
recordings were obtained in conventional whole cell patch-clamp
configuration with an Olympus IX70 inverted microscope, EPC-9
amplifier, and Pulse software (HEKA Electronics). The glass-
bottomed chambers with adherent cells were washed with PBS
and then filled with standard extracellular solution consisting of
140 mM NaCl, 2.8 mM KCl, 10 mM HEPES, 1 mM MgCl2,
2 mM CaCl2, 10 mM glucose, pH adjusted to 7.2–7.4, and
osmolarity adjusted to 300–310. The pipette solutions contained
10 mM NaCl, 145 mM cesium glutamate, 10 mM HEPES,
2 mM CaCl2, 1 mM MgCl2, 0.1 mM EGTA, 2 mM ATP,
0.3 mM GTP, titrated with 5 M CsOH to pH 7.2–7.4, and
osmolarity adjusted to 290–300.
Measurement of [Ca2+]iin response to glucose
In order to observe calcium changes associated with glucose
stimulation, we used the ratiometric calcium dye Fura2-AM
(Invitrogen). The intracellular Ca2+concentration was determined
by the ratio of the excitation of the ratiometric calcium dye Fura2-
AM (Invitrogen) at 350 and 380 nm using the Polychrom V by Till
Photonics. Emission was measured at 500–520 nm using a
Photometric Cascade CCD camera and Metafluor Software.
Cells were incubated at room temperature for 1 hour in
standard patch-clamp external solution with a modified glucose
concentration of 3 mM and supplemented with 4 mM Fura2-AM.
The cells were washed three times with 3 mM glucose external
solution before recording. Fura-2 fluorescence was detected with a
timelapse of 300 ms at 500–520 nm wavelength following
excitation at 350 nm (F350) and 380 nm (F380). The ratio,
F350/F380, was calculated after subtracting background from
each wavelength using Igor programming software. After
recording fluorescence measurements in 3 mM glucose, high-
glucose extracellular solution was added for a final concentration
of 14 mM glucose to stimulate glucose response. The ratio, F350/
F380, was again obtained using Igor software.
T15 cells by pEGFP-N1.
Transfection efficiency in MB-231 and HIT-
TC-6 cells with the indirect dual-color reporter. (a, b)
Confocal images of HIT-T15 (a) and beta-TC-6 (b) cells. Green
and red fluorescence is colocalized in one cell.
Transiently transfected HIT-T15 and beta-
The authors thank S. Wang, L. Zhang, D. Gibson, and Le Ma (USC) for
reagents, and C. Newgard (Duke University) for INS-1 832/13 cells. The
authors also thank other members of the Chow lab for assistance with using
Analyzed the data: NSL. Contributed to very helpful discussions/advice:
PMS KH MP AW RHC. Edited the manuscript: PMS KH MP AW RHC.
Executed experiments of constructing reporters with SK, and all other
molecular and cell biological assays: NSL. Performed electrophysiological
experiments: JGR. Contributed to measurement of [Ca2+]I: MZ.
Contributed to imaging of [Ca2+]I: AW. Contributed pINS-GFP
construct: KH. Helped write the manuscript: PMS. Designed and wrote
the manuscript: NSL RHC.
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