Limitations of Green Fluorescent Protein as a ... - Wiley Online Library

TECHNOLOGY DEVELOPMENT Limitations of Green Fluorescent Protein as a Cell Lineage Marker E. SCOTT SWENSON,a JOANNA G. PRICE,b TIMOTHY BRAZELTON,c DIANE S. KRAUSEb a

Section of Digestive Diseases, Department of Internal Medicine, and bDepartment of Laboratory Medicine, Yale University School of Medicine, New Haven, Connecticut, USA; cDepartment of Surgery, Children’s Hospital of Philadelphia, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA Key Words. Green fluorescent protein • Lineage tracing • Bone marrow transplantation • Donor marker

ABSTRACT The enhanced green fluorescent protein (GFP) reporter has been widely adopted for tracking cell lineage. Here, we compare three transgenic mouse strains in which GFP is considered “ubiquitously expressed,” with the GFP transgene under control of the chicken ␤-actin (CBA) or human ubiquitin C (UBC) promoter. We compared the expression of GFP using flow cytometry, direct tissue fluorescence, and immunostaining with multiple commercially available antiGFP antibodies. Mice of CBA-GFP strain 1Osb have strong but variegated expression of GFP in adult liver, kidney, small intestine, and blood. Mice of CBA-GFP strain Y01 have the highest proportion of GFP-positive peripheral blood cells yet limited GFP expression in liver, intestine, and kidney. UBC-GFP mice express GFP only weakly in solid organs and variably in blood. Direct fluorescent detection of GFP in for-

malin-fixed, paraffin-embedded tissue sections was the simplest approach, but it was useful only in high-expressing strains and potentially subject to artifact because of tissue autofluorescence. Immunofluorescence using either primary goat or primary rabbit antibodies was much more sensitive and allowed better discrimination of authentic signal from autofluorescence. Immunohistochemical staining was less sensitive than direct fluorescence or immunofluorescence and was subject to false-positive signal in the small intestine. In conclusion, there is considerable variability of expression within and between GFP transgenic strains. None of the tested strains gave truly ubiquitous GFP expression. A detailed analysis of GFP expression in one’s tissues of interest must guide the choice of reporter mouse strain when GFP is used as a marker of cell lineage or donor origin. STEM CELLS 2007;25:2593–2600

Disclosure of potential conflicts of interest is found at the end of this article.

INTRODUCTION Enhanced green fluorescent protein (GFP) has become the marker of choice for many types of cell transplantation and lineage marking experiments. It is readily detected in live, frozen, or fixed cells using established methods and is detected with high sensitivity and specificity in suspended cells by flow cytometry. The sensitivity and relative simplicity of GFP detection make it appealing. However, there are limitations of GFP as a marker (reviewed in [1]). For example, GFP may lose its direct fluorescence during tissue fixation or subsequent processing. For this reason, immunostaining with commercially antibodies is often used for GFP detection. In addition to these technical concerns, there can be considerable biological variability of GFP expression, even among similar cell types in a single animal [2]. Prior to conducting transplantation or lineage-marking experiments, it is essential to conduct a thorough analysis of the expression pattern of GFP in appropriate control animals. Several lines of “ubiquitously expressing” GFP transgenic mice are available, yet there can be tremendous variability at multiple levels of expression due to differences in absolute abundance of GFP and/or in the percentage of cells expressing GFP. High-level expression in the blood does not necessarily indicate high-level expression in solid organs. Even mice that appear to glow bright green under appropriate fluorescent light [3] may not be uniformly green at the level of individual cells.

Strains of GFP mice often have individuals with high, intermediate, or low expression and may be bred selectively for high or low expression [1]. However, high levels of GFP expression may have subtle toxic effects, so that over time, unselected transgenic lines may “drift” toward a relatively lower level of expression. Judicious selection of GFP transgenic strain and careful optimization of the detection method should improve experimental consistency. In this report, we compare three different transgenic lines of GFP-expressing mice by different detection methods.

MATERIALS

AND

METHODS

Mice All mice used in the study were sacrificed at 6 – 8 weeks of age. The chicken ␤-actin-enhanced GFP strain C57BL/6-TgN(ACTBEGFP)1Osb, originally developed by Dr. Masaru Okabe (Osaka University, Osaka, Japan) [3], was obtained from the Jackson Laboratory (Bar Harbor, ME, http://www.jax.org; stock no. 003291). This transgene consists of a chicken ␤-actin promoter, cytomegalovirus (CMV) enhancer, ␤-actin intron, and bovine globulin polyadenylation signal. The strain C57BL/6-TgN(ACTB-EGFP) OsbY01 (also developed by Dr. Okabe and obtained with permission from Dr. Okabe and Dr. William Fleming, The Ohio State University, Columbus, OH) represents the same transgenic construct as the initial strain, but it is integrated into a different chromosomal site [4]. The third GFP mouse strain analyzed,

Correspondence: E. Scott Swenson, M.D., Ph.D., Section of Digestive Diseases, Yale University School of Medicine, New Haven, Connecticut 06520-8019, USA. Telephone: 203-785-7054; Fax: 203-785-7095; e-mail: [email protected] Received March 30, 2007; accepted for publication June 17, 2007; first published online in STEM CELLS EXPRESS July 5, 2007. ©AlphaMed Press 1066-5099/2007/ $30.00/0 doi: 10.1634/stemcells.2007-0241

STEM CELLS 2007;25:2593–2600 www.StemCells.com

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Table 1. Antibodies Sourcea

Primary antibodies Rabbit pAb Goat pAb Mouse mAb Isotype controls Rabbit Goat Mouse Secondary antibodies Biotinylated goat anti-rabbit AlexaFluor 568 goat anti-rabbit AlexaFluor 568 donkey anti-goat Biotinylated donkey anti-goat IgG Biotinylated goat anti-mouse

Catalog number

Isotype

Invitrogen Abcam Clontech

A11122 6673 632381

IgG IgG IgG2a

Calbiochem Calbiochem DAKO

NI01 NI02 X0943

IgG IgG IgG2a

DAKO Invitrogen Invitrogen Jackson Immunoresearch Invitrogen

E0432 A11036 A11057 705-065-003 B-2763

a

Abcam, Cambridge, U.K., http://www.abcam.com; Calbiochem, San Diego, http://www.emdbiosciences.com; Clontech, Palo Alto, CA, http:// www.clontech.com; DAKO, Glostrup, Denmark, http://www.dako.com; Invitrogen, Carlsbad, CA, http://www.invitrogen.com; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com. Abbreviations: mAb, monoclonal antibody; pAb, polyclonal antibody.

C57BL/6 Tg(human ubiquitin C [UBC]-GFP)30Schaa/J (stock no. 004353; Jackson Laboratory), consists of GFP under the control of the human ubiquitin C promoter, an SV40 polyadenylation signal, and a bovine growth hormone polyadenylation signal [5]. Normal controls were age and sex-matched nontransgenic C57BL/6 mice from the Jackson Laboratory.

normalized to a peak intensity value of 100, and the corresponding background signal from GFP-negative tissue was normalized identically. Positive control tissue included fixed skeletal muscle from 1Osb GFP transgenic mice. Spectra of soluble GFP at pH 7 were obtained from Molecular Probes Inc. (Eugene, OR, http://probes. invitrogen.com).

Flow Cytometry

Immunohistochemistry

Blood was collected from the retroorbital plexus of anesthetized mice into heparinized capillary tubes. Following erythrocyte lysis in ammonium chloride (PharmLyse; BD Biosciences, San Diego, http://www.bdbiosciences.com), nucleated cells were washed and resuspended in phosphate-buffered saline (PBS) (pH 7.2) supplemented with 2% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com). The proportion of cells expressing GFP was determined by flow cytometry using appropriate nontransgenic controls for comparison.

Antigen retrieval was performed using Signet pH-All-2 (product no. 1922; Signet Laboratories, Dedham, MA, http://www.signetlabs. com) for 30 minutes in steam, and solution was then cooled to room temperature and washed in PBS. Endogenous peroxides were blocked using 3% H2O2 in PBS at room temperature for 10 minutes. Endogenous biotin was blocked using the Avidin/Biotin Blocking Kit (Vector Laboratories). Nonspecific binding of immunoglobulins was blocked by incubating slides in a 10% solution of normal serum from the species in which the secondary antibody was prepared (goat or donkey). The primary antibodies, isotype controls, and secondary antibodies used are indicated in Table 1. Primary antibodies and isotype controls were diluted in PBS supplemented with 1% BSA and applied to tissue sections at a final concentration of 10 ␮g/ml. Following overnight incubation at 4°C, slides were washed three times in PBS, and secondary antibody was applied at a final concentration of 10 ␮g/ml in PBS with 1% BSA for 1 hour at 37°C. Biotinylated secondary antibodies were applied at a final concentration of 10 ␮g/ml in PBS with 1% BSA for 1 hour at 37°C. Antibody complexes were detected using a streptavidinhorseradish peroxidase complex (Vectastain ABC Elite system; Vector Laboratories) and diaminobenzidine (DAB) substrate (Vector Laboratories). Slides were counterstained with hematoxylin (Vector Laboratories), cleared in ethanol, and permanently mounted (Permount; Fisher Scientific).

Tissue Collection and Processing Mice were treated humanely under a protocol approved by the Yale University Institutional Animal Care and Use Committee. Mice were sacrificed under deep anesthesia with ketamine and xylazine. Organs were quickly dissected into sections not more than 5 mm thick and then fixed in formalin for 4 hours at room temperature. Formalin was replaced by 70% ethanol until tissues were embedded in paraffin. Five-micrometer sections were prepared by the Research Histology Service at Yale University.

Direct Fluorescence Slides were heated to 60°C for 2 minutes, deparaffinized in Citrisolv (Fisher Scientific International, Fairlawn, NJ, http:// www.fisherscientific.com), and rehydrated through graded ethyl alcohols to PBS. Slides were rinsed in distilled water and air-dried. Vectashield with 4,6-diamidino-2-phenylindole (DAPI) (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and glass coverslips were applied. Slides were viewed under fluorescence microscopy using appropriate filters. Fluorescent emission spectra were acquired using a Leica SP2 confocal microscope (Heerbrugg, Switzerland, http://www.leica.com) equipped with three lasers (argon, 543 nm HeNe, and 633 nm HeNe) and three spectral detectors. Each excitation wavelength was used sequentially, and excitation wavelengths of 458, 488, 514, and 543 nm were selected with an acousto-optical tunable filter with subnanometer resolution. Putative GFP-positive and -negative areas were gated and imaged simultaneously using identical settings, and the mean emission intensity values for each area were measured in 2-nm increments (a ␭ series) throughout the relevant spectrum. Mean intensity values for the putative GFP-positive areas were

Immunofluorescence Slides were deparaffinized and antigen retrieval performed as described above, except that peroxidase and biotin blocking steps were omitted. Primary antibody was rabbit or goat polyclonal anti-GFP (Table 1). Appropriate rabbit or goat IgG isotype negative controls were run in parallel. Nontransgenic tissues served as additional negative controls. Fluorescent-conjugated secondary antibodies were goat anti-rabbit IgG AlexaFluor 568 (A11036; Molecular Probes) or donkey anti-goat IgG AlexaFluor 568 (A11057; Molecular Probes) applied at 10 ␮g/ml in PBS with 1% BSA for 1 hour at 37°C. Slides were washed in PBS, rinsed in water, and then air-dried. Fluorescence was visualized using Vectashield with DAPI (Vector Laboratories).

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Figure 1. GFP immunohistochemistry. Three commercially available antibodies were compared, using diaminobenzidine substrate (brown) for peroxidase. Liver sections were prepared from one mouse of the 1Osb strain and stained with rabbit primary (Molecular Probes), goat primary (Abcam), or mouse primary (Clontech) antibody (left column). Isotype controls for each primary antibody are shown in the center column. Nontransgenic controls are shown in the right column. Abbreviation: GFP, green fluorescent protein.

Photomicrographs Photomicrographs were taken using an Olympus BX51 fluorescence microscope (Tokyo, http://www.olympus-global.com) using bright-field illumination or appropriate fluorescence excitation and emission filters. Fluorescent images were captured with a digital camera, using IPLab software (BD Biosciences). Bright-field images were captured with QCapture Pro software (QImaging, Burnaby, BC, Canada, http://www.qimaging.com).

Statistical Analysis The percentage of GFP-positive blood cells was compared by the t test. A p value ⬍.05 was considered significant.

RESULTS Comparison of Anti-GFP Antibodies To clarify which antibody works best in paraffin sections, we first compared three commercially available anti-GFP antibodies (Table 1) on multiple tissues from mice of the 1Osb strain or nontransgenic controls, using horseradish peroxidase-DAB immunohistochemistry (Fig. 1; supplemental online Fig. 1). In each case, equivalent concentrations of isotype control antibody were run in parallel to test for nonspecific staining. As shown in Figure 1A, the rabbit polyclonal antibody to GFP stains a subset of hepatocytes in a mouse of the 1Osb strain, with little or no nonspecific staining in isotype (Fig. 1B) or nontransgenic (Fig. 1C) negative controls. The goat polyclonal anti-GFP antibody stains in a similar pattern (Fig. 1D), although with reduced intensity. The mouse monoclonal antibody also stains GFP-expressing hepatocytes in a similar pattern (Fig. 1G), but there was substantial nonspecific background staining seen in endothelial and Kupffer cells in the liver (Fig. 1H, 1I). www.StemCells.com

Immunohistochemical staining of GFP in the small intestine of the same mouse using rabbit, goat, or mouse primary antibody, compared with isotype and nontransgenic controls, is shown in supplemental online Figure 1. In the small intestine, a high rate of false-positive staining of cells in both the epithelium and lamina propria was seen with isotype (supplemental online Fig. 1B, 1E, 1H) and nontransgenic controls (supplemental online Fig. 1C, 1F, 1I) for rabbit, goat, or mouse primary antibody.

Comparison of GFP Detection Methods Direct detection of GFP fluorescence in formalin-fixed, paraffin-embedded sections has been regarded by some investigators as insensitive and unreliable because of rapid loss of soluble, cytoplasmic GFP from the tissue during fixation and processing. We found direct detection of GFP to be simple and sensitive in 1Osb mice, where the absolute level of GFP expression is high. We also noted that the intrinsic fluorescence of GFP is not eliminated by antigen retrieval in steam (Fig. 2B, 2E), a testament to the remarkable heat stability of GFP fluorescence in formalin-fixed paraffin sections. We immunostained for GFP with a secondary antibody conjugated to AlexaFluor 568 (Fig. 2C) to distinguish intrinsic fluorescence from immunofluorescence. In the liver of 1Osb mice, where GFP expression is high, the same variegated pattern was observed by direct GFP fluorescence, immunofluorescence, and immunohistochemistry (compare Fig. 2B vs. 2C and Fig. 2E vs. 2F). To confirm that the green signal seen by direct fluorescence was GFP rather than autofluorescence, we performed a spectral analysis (Fig. 3). Background red autofluorescence in the liver is shown in Figure 3A. The pattern is uniform and does not resemble the pattern seen by direct GFP fluorescence (Fig. 3B), indicating that the green fluorescence was not autofluorescence. To confirm that the bright green cells

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Figure 2. Comparison of direct fluorescence versus immunofluorescence or immunohistochemistry in liver. (A): DAPI staining of nuclei. (B): No antibodies were applied. The intrinsic fluorescence of GFP (green) was photographed directly after clearing away paraffin and performing antigen retrieval. This section was then stained with anti-GFP and a red (AlexaFluor 568) secondary antibody (C) to distinguish immunofluorescence from direct fluorescence. Immunofluorescence stained all the cells seen by direct fluorescence, as well as additional cells surrounding the large blood vessel (arrows). In a merged image (D), yellow reflects the combination of green (direct fluorescence) with red (immunofluorescence). Red cells are those detected only by immunofluorescence. (E): Another liver section was cleared of paraffin and photographed by direct fluorescence. This section was subsequently stained with anti-GFP and diaminobenzidine (DAB) immunohistochemistry (F). The same fluorescent green cells in (E) stained with DAB in (F), but the staining was less sensitive compared with immunofluorescence. Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFP, green fluorescent protein.

represented GFP rather than autofluorescence, clusters of 1Osb hepatocytes were visually categorized as GFP-bright or GFP-negative (Fig. 3C). These areas are indicated by red and yellow boundaries, respectively, in Figure 3C. Emission spectra from these areas were compared with each other and with soluble GFP protein or skeletal muscle from a 1Osb GFP transgenic mouse (Fig. 3D) to confirm that the clusters of bright green hepatocytes seen by direct fluorescence express bona fide GFP.

GFP Expression in Different Transgenic Lines GFP Expression in Peripheral Blood. GFP expression in peripheral blood was assessed in each strain of GFP mice by flow cytometry. Representative histograms for each strain are shown in the top row of Figure 4. Nontransgenic negative controls for each experiment are shown in the second row. Mice of the 1Osb (n ⫽ 5) or UBC-GFP (n ⫽ 3) strain expressed GFP in only 70%–75% of blood cells, whereas mice of the Y01 strain (n ⫽ 5) expressed GFP in 93% of nucleated peripheral blood cells, a significantly greater percentage than either 1Osb mice (p ⬍ .0001 vs. Y01) or UBC-GFP mice (p ⬍ .05 vs. Y01). GFP Expression in Organs. Using immunohistochemistry, GFP could not be reliably distinguished from background

in Y01 or UBC-GFP mice (data not shown). We found immunofluorescent detection using the rabbit or goat antiGFP antibodies to be the most sensitive and reliable method for liver, small intestine, and kidney. Antigen retrieval with the Signet pH-All-2 reagent was superior to standard citrate buffer or no antigen retrieval (data not shown). Therefore, we used immunofluorescence to compare GFP expression in paraffin sections of liver, small intestine, and kidney in the three transgenic strains and compared them with nontransgenic controls. Representative GFP immunofluorescence photomicrographs using the goat anti-GFP are shown in Figure 5. Similar results were obtained with the rabbit anti-GFP (not shown). In mice of the 1Osb strain, GFP expression was very strong in some hepatocytes yet absent in neighboring hepatocytes (Fig. 5A). Erythrocyte autofluorescence was seen in all liver samples (Fig. 5A–5C) and is readily distinguished from GFP-positive hepatocytes by the characteristic shape and intravascular location of erythrocytes. In the small intestine, GFP was highly, but not uniformly, expressed in differentiated villus enterocytes (Fig. 5D). Intestinal smooth muscle and Paneth cells in the crypts appeared to express GFP as well, but this interpretation was limited by a high level of background staining in smooth muscle and Paneth cells in the negative controls (Fig. 5E, 5F). In the kidney, GFP was highly expressed in glomeruli (Fig. 5G). Renal tubular cells


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Figure 3. Spectral analysis of green hepatocytes in 1Osb mouse liver. (A): Red autofluorescence is uniformly distributed. (B): Direct fluorescence in the GFP channel shows clear demarcation between GFP-positive and GFP-negative hepatocytes. (C): Clusters of putative GFP-positive cells (selected by intensity values after 488 nm excitation and filtration through a 500- to 540-nm band pass; highlighted in red) and GFP-negative cells (yellow) were subjected to spectral analysis across several excitation and emission wavelengths to generate the emission profiles shown in (D). (D): The shapes of emission profiles (i.e., spectral response) generated from putative GFP-positive cells closely match the emission profiles of GFP⫹ control tissue (fixed GFP⫹ skeletal muscle), as well as purified soluble GFP. In contrast, the putative GFP-negative areas failed to generate a GFP-like emission spectrum. Abbreviations: EX, excitation; GFP, green fluorescent protein.

Figure 4. Expression of GFP in blood. The expression of GFP in nucleated peripheral blood cells was quantitated by flow cytometry. Representative histograms for each transgenic line are shown, with negative controls for each shown below. GFP was detected in FL-1 (x-axis). Abbreviations: #, number; GFP, green fluorescent protein; pos, positive.

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Figure 5. Green fluorescent protein (GFP) expression by immunofluorescence in liver, small intestine, and kidney in the 1Osb strain. GFP was immunostained using the goat anti-GFP antibody and donkey antigoat AlexaFluor 568. Left column, goat primary antibody; center column, isotype controls; right column, nontransgenic negative controls stained with goat anti-GFP. Because of the strong persistence of intrinsic GFP fluorescence, the green channel was deliberately omitted from these photographs to show immunofluorescence alone.

(Fig. 5G) exhibited a variegated pattern of GFP expression, reminiscent of that seen in hepatocytes. Erythrocyte autofluorescence was also noted in the kidney negative controls (Fig. 5H, 5I). Comparison of the pattern of GFP expression in the three transgenic lines using immunofluorescence revealed additional differences (Fig. 6). In the 1Osb liver, GFP expression was very strong in some hepatocytes but weak or absent from their neighbors (Fig. 6A). In contrast, Y01 liver had strong GFP expression in bile ducts and Kupffer cells but not hepatocytes (Fig. 6B). The UBC liver expressed GFP so weakly (Fig. 6C) that it was essentially indistinguishable from nontransgenic controls (Fig. 6D). In the small intestine of 1Osb mice, GFP was expressed strongly in mature enterocytes at the villus tip and in Paneth cells at the base of the crypt but less strongly in lamina propria cells and smooth muscle (Fig. 6E). In Y01 small intestine, GFP expression was strong in villus enterocytes, lamina propria, Paneth cells, and smooth muscle (Fig. 6F). In UBC intestine, GFP was strong in mature villus enterocytes and smooth muscle but not Paneth cells or lamina propria (Fig. 6G). The nontransgenic intestine was effectively GFPnegative, although the small area of apparent red staining illustrates the potential for false-positive detection when a portion of the tissue section lifts or curls from the surface of the slide (Fig. 6H). In the kidney, a subpopulation of renal tubular cells in both 1Osb mice and Y01 mice expressed GFP strongly (Fig. 6I, 6J). In contrast, GFP was abundant in Y01 glomeruli but not 1Osb glomeruli. UBC kidney (Fig. 6K) was essentially indistinguishable from nontransgenic control (Fig. 6L). In addition to the interstrain comparison described above, we also examined the variability of GFP expression in organs among mice of the same transgenic strain. We found little variation among individual Y01 or UBC-GFP mice (data not shown) but marked variation among mice of the 1Osb strain,

confirming an earlier report [1]. Three representative 1Osb livers are shown in supplemental online Figure 2. The proportion of hepatocytes expressing GFP varied from approximately 80% to less than 50%. Similar variability was noted in the small intestine and kidney (not shown).

DISCUSSION GFP has understandably become a popular marker for biological lineage tracing and transplantation studies. To better interpret the results of lineage tracing experiments using GFP, we have undertaken a detailed analysis of widely available antibodies and GFP mice. In 1Osb or Y01 mice, GFP was readily detected in formalin-fixed tissue sections from liver, intestine, or kidney. We deliberately restricted our analysis to formalin-fixed paraffin sections, because they are markedly superior to frozen sections in preservation of tissue morphology. We found that immunofluorescent detection of GFP was more sensitive than DAB immunohistochemistry or direct fluorescence. In UBCGFP mice, GFP was seen in small intestine but was so weak in liver and kidney that it could not be reliably detected by any method we tested. It remains possible that GFP could be detected in frozen tissue sections from UBC-GFP mice. We found that the goat polyclonal antibody (Abcam) and the rabbit polyclonal antibody (Molecular Probes) performed well in liver and kidney. However, in the small intestine, all three primary anti-GFP antibodies tested caused nonspecific staining of villus enterocytes when detected with DAB, underscoring the critical importance of appropriate negative controls when using this method. Immunofluorescence reduced, but did not eliminate, this problem in the intestine. Among mice that strongly express GFP in epithelial cells, such as 1Osb mice, the variegation of expression in those cells can limit the sensitivity of detection of transplanted cells by a factor equivalent to the proportion of donor cells that do not


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Figure 6. GFP immunofluorescence in liver, small intestine, and kidney in the three transgenic strains and nontransgenic control. For each transgenic strain, one representative animal is shown. (A): GFP appears yellow in 1Osb liver because of the merge of green (direct fluorescence) and red (immunofluorescence) signals. GFP is seen as red signal in bile ducts and Kupffer cells in Y01 liver (B) but not in hepatocytes. GFP expression in UBC-GFP liver (C) was indistinguishable from nontransgenic liver (D). (E–H): GFP expression in small intestine of 1Osb (E), Y01 (F), UBC-GFP (G), or nontransgenic mice (H). (I–L): GFP expression in kidney. A variegated pattern of GFP expression was seen in renal tubules of 1Osb mice (I) and Y01 mice (J). GFP in UBC-GFP kidney (K) was indistinguishable from nontransgenic control (L). Arrow in (H) indicates false-positive staining on a villus that was folded upon itself during processing. Abbreviations: GFP, green fluorescent protein; UBC, human ubiquitin C.

express GFP. For epithelial cells of the liver, intestine, and kidney, this proportion may exceed 50% (Figs. 4 – 6). Enrichment of the donor population by fluorescence-activated cell sorting selection for GFP-expressing cells prior to transplantation might reduce this problem. The transgenic construct is identical between the Y01 mouse and the 1Osb mouse, so the site of transgene integration likely accounts for at least part of the difference in expression between these lines. A similar variegated pattern was seen in multiple organs of CMV-GFP mice [6]. The mechanism of silencing of GFP in tissues of the 1Osb mouse is unknown. In hematopoietic stem/progenitor cells of 1Osb mice, GFP expression is gradually lost with differentiation [7]. However, the murine stem cell vector retrovirus conferred long-term, stable expression of GFP, but not DsRed, in transfected mouse hematopoietic stem cells [8], indicating that hematopoietic differentiation alone does not account for GFP silencing. Silencing of GFP in lentiviral-transfected rat hepatoblasts has been reported when the GFP was driven from the CMV promoter/enhancer but not from the albumin promoter [9]. Therefore, one may speculate that cytosine methylation of CMV regulatory elements plays a role in GFP silencing in the 1Osb mouse. The phenomenon of in vivo somatic deletion of the toxic albumin-uroplasminogen activator transgene from mouse hepatocytes, followed by expansion and repopulation of the liver by revertant clones, provides a dramatic example of the proliferative and regenerative potential of hepatocytes [10]. However, if GFP toxicity leads to somatic deletion of the transgene, then one would expect to find large clusters of GFP-negative hepatocytes, www.StemCells.com

rather than the apparent stochastic arrangement we observed. Others have proposed that the practical problem of GFP silencing can be overcome by increasing detection sensitivity using a tyramide amplification protocol in frozen tissue sections [11]. Whether this approach will work with paraffin sections has not been reported. We did not specifically examine the effect of cellular injury on GFP expression in these mice, but we and others [12] have learned that many types of liver injury dramatically increase autofluorescence, potentially leading to false-positive GFP expression when assessed by direct fluorescence. This problem may account for the unusually high degree of apparent hepatocyte engraftment from transplanted GFP donor bone marrow after CCl4 injury [13] and provides further impetus for using immunofluorescence and/or confocal microscopy with variable excitation and emission filters to distinguish GFP from autofluorescence. The reported ability of bone marrow-derived stromal and hematopoietic stem cells to differentiate across traditional developmental boundaries has generated a great deal of interest and controversy [14 –16]. Widely different results of bone marrow “plasticity” experiments are likely due not only to differences in experimental design but also to our limited understanding of the molecular events underlying the required alterations in the cell’s gene expression pattern, described as nuclear reprogramming. Nuclear reprogramming might be induced by local cues in the microenvironment or could occur through cell-cell fusion, as has been shown in the fumarylacetoacetate hydrolase knockout mouse after transplantation of wild-type


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bone marrow [17, 18]. It is not clear that GFP expressed in bone marrow stem cells or mature blood cells would necessarily continue to be expressed in epithelial or other marrow-derived cells after nuclear reprogramming, whether or not the reprogramming occurs by cell fusion. In the case of the Y01 GFP mouse strain, high expression of GFP in blood cells was not matched by high expression in hepatocytes. Thus, it would not be surprising that GFP or any other transgenic reporter would underestimate the frequency of nuclear reprogramming events. Genotypic markers such as the Y chromosome are not subject to such vagaries of transgene expression, indicating that genotypic markers rather than reporters, such as GFP, may be the preferred markers of donor cell origin in cell transplantation experiments. In this report, we have investigated the assumption that “ubiquitous” GFP mice express GFP at high levels in every cell and pointed out that expression of GFP in blood cells does not predict expression of GFP in tissues, even when a “housekeeping” promoter such as ␤-actin is used. GFP remains a valuable reporter for many biological applications, but a

thorough analysis of its expression in control tissues should be undertaken prior to executing transplantation or lineagetracking experiments. The optimal method of GFP detection depends on the tissue of interest and must be accompanied by appropriate controls.

ACKNOWLEDGMENTS This work was supported by NIH Grants DK61846, HL073742, and DK073404 and by the Yale Center of Excellence in Molecular Hematology.

DISCLOSURE

The authors indicate no potential conflicts of interest.

REFERENCES 10 1 2 3 4 5 6 7 8

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Brazelton TR, Blau HM. Optimizing techniques for tracking transplanted stem cells in vivo. STEM CELLS 2005;23:1251–1265. Anderson DA, Wu Y, Jiang S et al. Donor marker infidelity in transgenic hematopoietic stem cells. STEM CELLS 2005;23:638 – 643. Okabe M, Ikawa M, Kominami K et al. ‘Green mice’ as a source of ubiquitous green cells. FEBS Lett 1997;407:313–319. Nakanishi T, Kuroiwa A, Yamada S et al. FISH analysis of 142 EGFP transgene integration sites into the mouse genome. Genomics 2002;80: 564 –574. Schaefer BC, Schaefer ML, Kappler JW et al. Observation of antigendependent CD8⫹ T-cell/dendritic cell interactions in vivo. Cell Immunol 2001;214:110 –122. Walter I, Fleischmann M, Klein D et al. Rapid and sensitive detection of enhanced green fluorescent protein expression in paraffin sections by confocal laser scanning microscopy. Histochem J 2000;32:99 –103. Van Overstraeten-Schlogel N, Delgaudine M, Beguin Y et al. Limitations of the use of GFP transgenic mice in bone marrow transplantation studies. Leuk Lymphoma 2006;47:1392–1393. Tao W, Evans BG, Yao J et al. Enhanced green fluorescent protein is a nearly ideal long-term expression tracer for hematopoietic stem cells, whereas DsRed-express fluorescent protein is not. STEM CELLS 2007; 25:670 – 678. Oertel M, Rosencrantz R, Chen YQ et al. Repopulation of rat liver by

POTENTIAL CONFLICTS OF INTEREST

OF

11 12 13 14 15 16 17 18

fetal hepatoblasts and adult hepatocytes transduced ex vivo with lentiviral vectors. Hepatology 2003;37:994 –1005. Sandgren EP, Palmiter RD, Heckel JL et al. Complete hepatic regeneration after somatic deletion of an albumin-plasminogen activator transgene. Cell 1991;66:245–256. Toth ZE, Shahar T, Leker R et al. Sensitive detection of GFP utilizing tyramide signal amplification to overcome gene silencing. Exp Cell Res 2007;313:1943–1950. McTaggart RA, Feng S. An uncomfortable silence em leader while we all search for a better reporter gene in adult stem cell biology. Hepatology 2004;39:1143–1146. Terai S, Sakaida I, Yamamoto N et al. An in vivo model for monitoring trans-differentiation of bone marrow cells into functional hepatocytes. J Biochem (Tokyo) 2003;134:551–558. Wagers AJ, Sherwood RI, Christensen JL et al. Little evidence for developmental plasticity of adult hematopoietic stem cells. Science 2002; 297:2256 –2259. Krause DS, Theise ND, Collector MI et al. Multi-organ, multi-lineage engraftment by a single bone marrow-derived stem cell. Cell 2001;105: 369 –377. Jang YY, Collector MI, Baylin SB et al. Hematopoietic stem cells convert into liver cells within days without fusion. Nat Cell Biol 2004; 6:532–539. Vassilopoulos G, Wang PR, Russell DW. Transplanted bone marrow regenerates liver by cell fusion. Nature 2003;422:901–904. Wang X, Willenbring H, Akkari Y et al. Cell fusion is the principal source of bone-marrow-derived hepatocytes. Nature 2003;422:897–901.

See www.StemCells.com for supplemental material available online.