AJP-Endo Articles in PresS. Published on September 17, 2002 as DOI 10.1152/ajpendo.00321.2002
1 Transgenic Mice with Green Fluorescent Protein-labeled Pancreatic -cells
Manami Hara,1 Xiaoyu Wang,2 Toshihiko Kawamura,3 Vytas P. Bindokas,4 Restituto F. Dizon,1
Sergio Y. Alcoser,5 Mark A. Magnuson,6 and Graeme I. Bell1,2,5
From the 1Department of Medicine, the 2Howard Hughes Medical Institute, the 3Department of Pathology, the 4Department of Neurobiology, Pharmacology and Physiology, and the 5
Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago,
Illinois 60637; and the 6Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee 37232. Address correspondence and reprint requests to Dr. Manami Hara, Howard Hughes Medical Institute Research Laboratories, The University of Chicago, 5841 South Maryland Avenue, MC1028, Chicago, IL 60637. Tel: (773) 702-9118. Fax: (773) 702-9237. Email:
[email protected]. Abbreviations: AM, acetoxymethyl; EGFP, enhanced-GFP; GFP, green fluorescent protein; MIP, mouse insulin I promoter; FACS, fluorescence-activated cell sorter; PCR, polymerase chain reaction
Running head: MIP-GFP transgenic mice
Copyright 2002 by the American Physiological Society.
2 ABSTRACT We have generated transgenic mice that express green fluorescent protein (GFP) under the control of the mouse insulin I gene promoter (MIP). The MIP-GFP mice develop normally and are indistinguishable from control animals with respect to glucose tolerance and pancreatic insulin content. Histological studies showed that the MIP-GFP mice had normal islet architecture with co-expression of insulin and GFP in the β-cells of all islets. We observed GFP expression in islets from embryonic E13.5 day through adult. Studies of β-cell function revealed no difference in glucose-induced intracellular calcium mobilization between islets from transgenic and control animals. We prepared single-cell suspensions from both isolated islets and whole pancreas from MIP-GFP transgenic mice and sorted the β-cells by fluorescence-activated cell sorting based on their green fluorescence. These studies showed that 2.4±0.2% (n=6) of the cells in the pancreas of newborn (P1) and 0.9±0.1% (n=5) of 8-week old mice were β-cells. The MIP-GFP transgenic mice may be a useful tool for studying β-cell biology in normal and diabetic animals.
Key word: insulin, diabetes, GFP, transgenic, β-cell
3 INTRODUCTION The insulin-producing β-cell of the pancreas plays a central role in the pathophysiology of diabetes mellitus with anatomical and functional loss of these cells leading to type 1 and type 2 diabetes mellitus, respectively (1). The identification and characterization of embryonic or adult stem cells that give rise to the β-cell could lead to cellular-based therapies for treating both forms of diabetes (2-4). Previous studies have shown that green fluorescent protein (GFP) from the jellyfish Aequorea victoria and its yellow and cyan derivatives could be utilized as reporter genes to label specific cell types including pancreatic β-cells by expressing GFP under the control of a tissue-specific promoter (5-12). One advantage of these proteins is that they can be detected in living cells since they fluoresce brightly upon exposure to ultraviolet light or blue light without the addition of coactivators or exogenous substrate. In addition, pure populations of fluorescent-tagged cells can be isolated using a fluorescence-activated cell sorter (FACS) (9, 10, 12). Studies of rat and human β-cells treated with recombinant adenovirus expressing green fluorescent protein (GFP) under the control of the rat insulin I promoter appear to function normally suggesting expression of GFP may be well-tolerated by these cells (11, 12). Thus, we reasoned that if we could generate a mouse model in which we had genetically tagged the pancreatic β-cells with GFP, we would have a potentially valuable research tool for studying βcell biology including the identification of progenitor cells. Here, we describe a line of transgenic mice in which the pancreatic β-cells are genetically tagged with GFP.
4 MATERIALS AND METHODS Generation of MIP-GFP transgenic mice. The mouse insulin I (MIP) promoter-GFP transgenic construct was assembled using a 8.5 kb fragment of the mouse insulin I promoter that includes a region from -8.5 kb to +12 bp (relative to the transcriptional start site), the coding region of enhanced GFP (EGFP) (0.76 kb) (Clontech, Palo Alto, CA), and a 2.1 kb fragment of the human growth hormone cassette gene (hGH) for high-level expression (13, 14). The 11.2 kb MIPEGFP-hGH fragment was isolated from the vector by digestion of the plasmid construct with Sfi I and Hind III and agarose gel electrophoresis. The fragment was further purified using an Elutip-D column (Schleicher & Schuell, Keene, NH). The purified transgene DNA was microinjected into the pronuclei of CD-1 mice by the Transgenic Mouse/ES Core Facility of the University of Chicago Diabetes Research and Training Center (DRTC). Tail DNA from potential founder mice was screened for the presence of the transgene by PCR using forward and reverse primers 5’-GAAGACAATAGCAGGCATGCTG-3’ and 5’ACTGGGCTTACATGGCGATACTC-3’, respectively. All the procedures involving mice were approved by the University of Chicago Institutional Animal Care and Use Committee. Glucose tolerance testing. Intraperitoneal glucose tolerance tests (IPGTTs) were performed after a 4-h fast. Blood was sampled from the tail vein before and 30, 60, 90 and 120 min after intraperitoneal injection of 2 mg/g (body weight) of dextrose. Glucose levels were measured using a Precision Q.I.D.TM Glucometer (MediSense, Waltham, MA). Insulin assays. Pancreatic insulin content was measured after acid ethanol extraction of the whole pancreas as described previously (15). Insulin concentration was measured by a doubleantibody radioimmunoassay using a rat insulin standard in the Radioimmunoassay Core
5 Laboratory of the University of Chicago DRTC. The intraassay coefficient of variation for this assay is 7%. All samples were assayed in duplicate. Isolation of pancreatic islets of Langerhans. Pancreatic islets were isolated using a modification of the procedure originally described by Lacy and Kostianovsky (16). Briefly, the pancreas was inflated with a solution containing 0.3 mg/ml collagenase (Type XI; Sigma, St. Louis, MO) in Hanks’ balanced salt solution, injected via the pancreatic duct. The inflated pancreas was removed, incubated at 37°C for 10 min, and shaken vigorously to disrupt the tissue. Following differential centrifugation through a Ficoll gradient to separate islets from acinar tissue, the islets were washed and then handpicked. They were plated on 12- or 35-mm coverslips to facilitate adherence for subsequent measurement of intracellular calcium or confocal microscopic visualization. The islets were cultured in RPMI 1640 supplemented with 10% (vol/vol) fetal bovine serum (FBS), 100 U/ml penicillin and 100 µg/ml streptomycin in a humidified incubator at 37°C in 95% air and 5% CO2. Preparation of single-cell suspensions from islets and pancreas. Isolated islets were incubated in a solution of 0.05% trypsin-EDTA (GIBCO, Grand Island, NY) at 37°C for 3 min. The digestion was stopped by adding RPMI 1640 with 10% (vol/vol) FBS. The pancreata from P1 mice were removed and digested in a solution containing 0.3 mg/ml collagenase, and then in 0.05% trypsin-EDTA. The resulting single cells were washed with PBS, resuspended in cold PBS with 10% (vol/vol) FBS and filtered using 70 µm mesh. Cells were stained with trypan blue to check viability and preparations showing more than 95% live cells were analyzed. The pancreatic cell suspension was diluted to 2x106 cells/ml with the PBS/FBS solution and then fixed in 4% paraformaldehyde.
6 Pancreatic islet morphology. The pancreas was removed, embedded in optimum cutting temperature compound (Tissue-Tek O.T.C., Sakura Finetek, Torrance, CA) and frozen in isopentane at -70°C. Serial sections were cut at 6 µm in thickness and fixed in 4% paraformaldehyde. GFP fluorescence is well retained under these conditions (15). The sections were stained with a polyclonal guinea pig anti-porcine insulin antibody to identify β-cells and with polyclonal rabbit anti-human glucagon, somatostatin and pancreatic polypeptide antibodies to identify α, δ and PP cells, respectively (DAKO Corp., Carpinteria, CA). Sections were also stained with a monoclonal anti-mouse GFP antibody (Clontech) to detect GFP expression and with a polyclonal goat anti-human growth hormone antibody (DAKO) to test for expression of growth hormone from the transgene construct. The primary antibodies were detected using Texas Red-conjugated anti-guinea pig/rabbit/goat IgG (H+L) and biotin-streptavidin-conjugated antimouse IgG (H+L) followed by Texas Red-conjugated streptavidin (Jackson ImmunoResearch Lab., West Grove, PA). Microscopic images were taken with a Nikon Eclipse E800 microscope with PCM-2000 (Nikon, New York, NY) and an Olympus SZX-RFL3 microscope (Olympus, Melville, NY). Intracellular calcium measurements. The GFP expression interferes with Fura-2 signals and as a consequence the calcium indicator Fura-2 cannot be used to monitor changes in intracellular calcium in GFP-expressing β-cells. The short wavelength excitation of the EGFP excitation spectrum extends down to ~350 nm which is in the range of Fura-2. Thus, excitation of Fura-2 will excite EGFP and contaminate the common emission spectrum. Fura Red is a calcium indicator with a large stokes shift, thereby minimizing the contamination of the calcium signal by GFP (18, 19). Fura Red has been successfully used in combination with GFP expression in other studies (20, 21); however, there are reports of some signal crosstalk when used with EGFP (22).
7 Another calcium indicator, X-rhod-1 which has an excitation/emission maxima of ~580/ 602 nm may also give a good separation of the signals from GFP and the calcium indicator (22); however, care must be taken, since X-rhod-1 has a certain selectivity for mitochondria (23). We used Fura Red in the studies described here. The isolated islets were loaded with 5 µmol/l Fura Red-AM (acetoxymethyl ester) (Molecular Probes, Eugene, OR) and changes in intracellular calcium were monitored using a Fluoview scanning laser confocal with an inverted IX70 microscope (Olympus). Tiempo real-time acquisition software (Olympus) was used to collect and plot the data. The Cy5 filter set (700/75 nm bandpass filter set) was used to detect Fura Red signals and to eliminate the contamination with GFP signals. Solutions were perfused in a temperature-controlled chamber using a TC-344 Dual Heater Controller (Warner Instrument Corp., Hamden, CT). All the measurements were performed at 34-35°C. Flow cytometric analysis. Flow cytometric analysis of GFP-labeled β-cells was carried out using a FACScan flow cytometer with Cell Quest software (Becton Dickinson, Franklin Lakes, NJ). GFP-expressing cells were detected using the FL1 channel (absorption spectra 530/30 nm). We have sorted both fixed and non-fixed cells. However, the studies described here were carried out using cells fixed as described above. Statistical analysis. Results are expressed as mean ± SEM. We compared groups using Anova (StatView software, SAS Institute Inc., Cary, NC). Differences were considered to be significant at P< 0.05.
8 RESULTS Expression of GFP in -cells. The MIP-GFP construct was injected into the fertilized eggs of CD-1 mice. We obtained three founders: one female (6504) and two males (6502 and 6508). GFP was expressed in the islets of all three founders. The founders 6502 and 6508 were estimated to have one copy of the transgene by quantitative real time PCR. The founder 6504 was estimated to have five copies and two of her male F1 pups, 6729 and 6719, each had one copy of the transgene. Progeny from transgenic lines 6502 and 6719 showed delayed recovery on IPGTT and non-uniform expression of GFP within their islets and were not studied further. The studies described here were carried out on line 6729 (Tg(MIP-GFP)6729Hara). The transgene has been maintained on the CD-1 background and mice have been housed under specific pathogen-free (SPF) conditions with free access to food and water. The pancreatic islets from the MIP-GFP transgenic animals were green when exposed to ultraviolet light (Fig. 1A). The overall islet architecture of the MIP-GFP mice was normal. The patterns of GFP and insulin expression within the islet were identical and GFP expression was only observed in β-cells (Fig. 1). There was no evidence of expression of GFP in non-β-cells of the islet or in the exocrine pancreas (Fig. 1D). A stacked confocal microscopic image of an isolated islet suggested that the steady state levels of GFP were similar in all β-cells (Fig. 1E). We also tested for expression of GFP in the pancreas and other tissues at 6-wk of age by Western blotting. We detected GFP expression only in pancreas and not in any of the other tissues examined: brain, fat, muscle, small intestine, spleen, heart, kidney, liver, uterus and testis (data not shown). In addition, we also examined sections from each of these tissues for the presence of isolated GFP-expressing cells and found none. The transgene construct includes the human growth hormone gene at its 3’-end since previous studies have shown that the presence of
9 heterologous introns provided by the human growth hormone gene resulted in enhanced expression of the transgene (13). Since there is no internal ribosomal entry site upstream of the growth hormone gene, there should be no growth hormone expression and as expected, we found no evidence of growth hormone in islets from MIP-GFP mice by immunohistochemical staining using sections from pituitary as a positive control (data not shown). Expression of the MIP-GFP transgene appears to be restricted to β-cells. The mouse insulin I promoter fragment that we used appears to be more tissue-specific in its expression than shorter fragments (that are more typically used to make insulin promoter driven fusion genes), since no expression was observed in brain using this construct whereas expression in the brain is often observed with the shorter promoter construct (14). Physiological characterization of MIP-GFP mice. The MIP-GFP mice developed normally. At 6-wk of age, there were no significant differences in body weight, fasting blood glucose and pancreatic insulin content between transgenic and nontransgenic CD-1 male mice (data not shown). Intraperitoneal glucose tolerance testing (IPGTT) at 6-wk showed no statistically significant difference in the response between transgenic and nontransgenic male animals (Fig. 2). We have followed body weight and performed IPGTTs on the transgenic mice up to 40-wk, body weight and non-fasting blood glucose levels up to 60-wk and none of the animals have shown any evidence of abnormal weight or hypo- or hyperglycemia (data not shown.) Glucose responsiveness of pancreatic islets from the MIP-GFP mice. We examined the effects of glucose on intracellular calcium mobilization in the islets of the MIP-GFP mice as a measure of β-cell function. We used Fura Red (24) to monitor intracellular calcium levels to allow measurement of signals from the calcium indicator in the presence of GFP. The islets from both MIP-GFP and nontransgenic mice exhibited a robust mobilization of calcium in response to
10 10 mM glucose with no apparent difference between the islets from transgenic and nontransgenic animals (Fig. 3). Similar responses were observed in response to 20 mM glucose and 50 mM KCl occasionally accompanied by oscillations in calcium levels (data not shown). FACS analysis of GFP-labeled -cells. The islets isolated from MIP-GFP mice (8-wk old) were dissociated into single cells. The GFP-labeled β-cells could be readily separated from the non-βcells cells by FACS (Fig. 4 shows a representative trace). Measurement of pancreatic -cell number. A single-cell suspension was prepared from whole
pancreas of a MIP-GFP transgenic mouse at P1 (Fig. 5A) and the percentage of pancreatic βcells was measured by FACS. Flow cytometric analysis revealed that 2.6% of the pancreatic cells expressed GFP (Fig. 5B). Further analyses on five additional transgenic littermates gave similar results and indicate that 2.4±0.2% (n=6) of the cells in the P1 pancreas were GFP-expressing βcells (each trace not shown). We have also prepared single-cell suspensions from whole pancreas of older mice and sorted the cells based on GFP expression. We found 0.9±0.1% (n=5) of the cells in pancreas of 8-wk old animals were GFP-positive (data not shown). Expression of MIP-GFP transgene during development. The pancreas begins to form at the 26 somite stage (E9.5) of gestation in the mouse (25). Although insulin mRNA can be detected at the 20-somite stage (25), significant expression of insulin only begins at E13.5 (26). We observed GFP-labeled cells in the pancreas at E13.5 (we did not examine earlier stages) and continuing throughout life. At E13.5, there is a scattered mass of GFP-expressing cells in the pancreas adjacent to the duodenum. GFP-labeled islets are evident at E18.5 and in the adult (60wk) where they can be readily seen distributed throughout the pancreas.
11 DISCUSSION We have generated transgenic mice in which pancreatic β-cells are genetically tagged with GFP. The phenotypic characterization of the MIP-GFP transgenic mice suggests that the presence of cytoplasmic GFP does not impair β-cell development or function, at least in the line of mice described in this report. We can envisage a number of uses of these mice for studying βcells in situ and in isolation. In this regard, β-cell function can be studied in the MIP-GFP mice or in intercrosses with other genetically engineered and mutant mice. We believe the MIP-GFP mice will be useful for isolating pancreatic β-cells at various stages of development from embryonic to adult. The purified β-cells can be used for molecular biological studies such as monitoring the changing pattern of gene expression during development using microarrays (27) or for biophysical studies of their functional properties (28). The isolation of a pure population of β-cells is arduous. Pipeleers and his colleagues have described a procedure that works well with rat islets beginning with purified islets (29). The islets are dissociated into single cells from which a highly enriched β-cell population can be obtained by FACS based on the high intrinsic autofluorescence of β-cells. Meyer et al. have described another procedure in which dispersed islet cells (in this case human) were infected with a recombinant adenovirus expressing GFP driven by the rat insulin I promoter (12). The GFP is only expressed in β-cells and these cells can be sorted by FACS to obtain a pure (>95%) population of β-cells. This procedure is generally applicable to isolating β-cells from many different species including mice and at various stages of development. However, special precautions must be taken when working with adenovirus, even the attenuated vectors that are commonly used in molecular biology. In contrast to these procedures, purified β-cells can be readily isolated from MIP-GFP mice beginning with either islets or pancreas. The ability to flow sort dissociated pancreatic cells to isolate will facilitate
12 studies of embryonic β-cells where islet isolation is very difficult. A second use of the MIP-GFP transgenic mice is in studies where real-time instant identification of β-cells is required such as in electrophysiological studies as the β-cells can be distinguished from non-β-cells based on their green fluorescence. Another use is in studies of β-cell development. β-cell progenitors and/or stem cells have been described in ductal tissue (30), adult marrow (4) and embryonic stem cells (2, 3) and the MIP-GFP mice may be useful in identifying the progenitor/stem cells in these tissues and cells. The preliminary data that we have presented here indicate that we can use FACS to quantify the number of β-cells in the pancreas and this may be a useful method for following the changes in β-cell number that occurs in different physiological states such as pregnancy and diabetes. We also believe that we may be able to use FACS analysis to monitor changes in β-cell size. Finally, the GFP fluorescence is sufficiently intense, especially in the adult, to be detected within a thick specimen including a whole pancreas (Fig. 5). Thus, it may be possible to carry out a 3-dimensional reconstruction of the distribution of islets within the entire pancreas of the MIP-GFP mice. In summary, we have generated a line of mice in which the β-cells are genetically tagged with GFP. We believe that the MIP-GFP mice will be a useful tool for studying β-cells and islets in normal and diabetic states.
ACKNOWLEDGMENTS This research was supported by U.S. Public Health Service Grants DK-20595, DK-44840 and DK-61245. G.I.B. is an Investigator of the Howard Hughes Medical Institute.
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17 Figure Legends
FIG. 1. Pancreatic β-cell specific expression of GFP. A: A confocal microscopic image of a pancreatic section shows GFP expression (green) in the central core of the islet. Scale bar is 50 µm. B: The same section was stained with an anti-insulin antibody (red). C: A dual-color image showed overlapping expression of GFP and insulin in the β-cells of the islet (yellow). D: A pancreatic section was stained with cocktail of antibodies against three non-β-cell hormones, i.e. glucagon, somatostatin and pancreatic polypeptide (red). There was no overlapping expression of GFP with other endocrine hormones. Scale bar is 50 µm. E: An isolated pancreatic islet from a MIP-GFP transgenic mouse. A stacked confocal microscopic image is shown. The dark region within each cell is the nucleus. Scale bar is 10 µm.
FIG. 2. Intraperitoneal glucose tolerance testing in 6-wk male MIP-GFP transgenic (MIP-GFP) and age and sex-matched wild-type (WT) mice. There was no significant difference in blood glucose levels at each time point (P>0.05).
FIG. 3. Pancreatic islets from the MIP-GFP mice retain normal glucose responsiveness. A: Representative trace of Fura Red-AM intensity showing the intracellular calcium increase in response to 10 mM glucose in cells within an isolated islet from a wild-type mouse. Note that fluorescence of the Fura Red decreases once the indicator binds calcium. The duration of administration is indicated by the horizontal bar above the traces. Fura Red-AM fluorescence was measured by drawing a region of interest around a group of the calcium indicator-loaded cells and plotted versus time. Fluorescence intensity is expressed in arbitrary units. B: GFP-
18 expressing β-cells show a similar response to 10 mM glucose. Inset: Merged confocal images of an isolated islet from a MIP-GFP mouse showing GFP-expressing β-cells (green) and peripheral cells that were loaded with Fura Red-AM (5 µM) (red) which appear yellow (green + red) demonstrating colocalization of indicator and GFP in β-cells. Scale bar is 20 µm.
FIG. 4. Flow cytometric analysis of a single-cell suspension prepared from islets from MIP-GFP transgenic and wild-type mice. Fluorescence intensity is expressed in arbitrary units on a logarithmic scale. The range of negative signals (autofluorescense) was determined using wildtype β-cells as a control. In the cell suspension prepared from isolated pancreatic islets from MIP-GFP transgenic mice (8-wk old), 90% of the cells fluoresce green.
FIG. 5. Flow cytometric analysis of a single-cell suspension prepared from whole pancreas of a P1 MIP-GFP transgenic mouse. A: Bright field image of a pancreatic cell suspension from a MIP-GFP transgenic mouse (left panel). An arrow in the left panel indicates GFP-expressing βcells that fluoresce green in the fluorescent image (right panel). The dissociated cells were concentrated by a brief centrifugation prior to viewing. Scale bar is 50 µm. B: Representative flow cytometric analysis of a single-cell suspension prepared from a wild-type mouse (left panel) and a MIP-GFP mouse (right panel). 2.6% of the pancreatic cells from the MIP-GFP mouse at P1 express GFP.
FIG. 6. GFP-expression in the pancreas from MIP-GFP transgenic mice at different developmental stages. A: Dark field image of an embryo at E13.5. Scale bar is 500 µm. B: Dark field image of the gut region including duodenum and pancreas from the embryo in A. An
19 enlarged image of the boxed area is shown in C. Scale bar is 500 µm. C: Fluorescent image of the pancreas (boxed area in B) showing GFP-labeled green β-cells. Scale bar is 100 µm. D: Dark field image of the gut from an embryo at E18.5. Scale bar is 500 µm. E: Fluorescent image of the gut in D. Note the mass of GFP-expressing cells scattered throughout the pancreas. F: Dark field image of gut from a mouse at birth (P0). Scale bar is 500 µm. G: Fluorescent image of the gut in F. H: Dark field image of the pancreas including spleen from a 60-wk old male mouse. Scale bar is 5 mm. I: Fluorescent image of the gut in H. Note the islets are readily evident as clusters of green cells dispersed throughout the pancreas.
