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Originally published online as doi:10.1189/jlb.0307141 on August 17, 2007

Published online before print August 17, 2007
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(Journal of Leukocyte Biology. 2007;82:1247-1256.)
© 2007 by Society for Leukocyte Biology

A hCXCR1 transgenic mouse model containing a conditional color-switching system for imaging of hCXCL8/IL-8 functions in vivo

Lei Zheng, Ching-ni Njauw and Manuela Martins-Green1

Department of Cell Biology and Neurosciences, University of California, Riverside, Riverside, California, USA

1 Correspondence: Department of Cell Biology and Neuroscience, University of California, Riverside, Riverside, CA 92521, USA. E-mail: manuela.martins{at}ucr.edu


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ABSTRACT
 
To address the functions of human CXCL8 (hCXCL8)/IL-8 through hCXCR1 in vivo, we have developed a humanized, transgenic mouse for hCXCR1. This mouse line is versatile and allows for a variety of functional analyses using bioimaging, including Cre/loxP-mediated, tissue-specific hCXCR1 expression in a spatiotemporal manner; a color-switching mechanism, which uses spectrum-complementary, genetically encoded green and red fluorescence markers to label the hCXCR1-expressing cells [enhanced GFP (eGFP)] against the background [monomeric red fluorescent protein (mRFP)]; a bioluminescent marker, which is present in the hCXCR1-expressing cells; and an exogenous cell surface marker (eGFP moiety) in the hCXCR1-expressing cells, which facilitates identification, isolation, and targeting of these cells. The established, transgenic founder line RCLG3A (TG+) expresses only mRFP and does so ubiquitously. When the RCLG3A mice are crossed with the tamoxifen-inducible, whole-tissue Cre mice (ROSA26-Cre/Esr+/–), administration of tamoxifen induces whole-body hCXCR1 expression and color-switching. When RCLG3A mice are crossed with thymocyte-specific Cre mice (Lck-Cre+/+), the hCXCR1 expression and color-switching are restricted in a lineage-specific manner. This mouse line can be used to understand the functions of hCXCL-8 in vivo. In addition, our approach and vectors can be used to establish other tissue-specific, transgenic mice in conjunction with multifunctional cell markers, which facilitate cell imaging, tracing, and manipulation in vivo.

Key Words: Cre/loxP • dual fluorescence • bioluminescence


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INTRODUCTION
 
Numerous pathological conditions such as autoimmune diseases, xenograft rejection, rheumatoid arthritis, atherosclerosis, and Alzheimer’s disease have inflammation as an important component [1 2 3 4 ]. Other conditions, such as Parkinson’s disease and age-related macular degeneration, are also associated with inflammation, suggesting that their pathogenesis may also be linked to an inflammatory process [5 , 6 ]. Much research has been performed to better understand the pathology of diseases associated with inflammation. As part of this effort, it has been discovered that chemokines play important roles in these diseases. Chemokines are a large family of small, secreted proteins (8–15 kD), which recruit various types of leukocytes [7 ]. It is now clear that these proinflammatory proteins also play a role in driving the maturation, homing, and activation of leukocytes, as well as the migration and function of other resident cell types associated with inflammatory processes. Moreover, a growing body of experimental evidence supports direct and pivotal roles for chemokines and their receptors in angiogenesis, wound healing, viral infections [8 ], and tumorigenesis [9 10 11 ].

Chemokines are classified into four distinct families, CXC, CC, C, and CX3C, based on the position of the first two conserved cysteines [12 ]. IL-8 (now referred to as CXCL8), serves as the prototype CXC chemokine, and although it has been studied for more than 10 years, the full repertoire of its functions in humans remains to be elucidated. IL-8 was discovered originally as a neutrophil chemoattractant [13 ], but it is known today that this chemokine is highly expressed in response to injury, be it a wound, a tumor, or a microbe. More recent work has shown that CXCL8 can also stimulate many different cell types to migrate, divide, and differentiate, depending on local cues [9 , 11 ]. For example, our work and that of others [14 15 16 ] has shown that CXCL8 stimulates angiogenesis and promotes wound closure and contraction. The latter effects result from fibroblast differentiation into myofibroblasts following stimulation by CXCL8 [15 ]. Furthermore, CXCL8 has been linked to a wide variety of human cancers; more than 15 types of human cancers and tumor-derived cell lines have been found to express high levels of CXCL8, and this chemokine has also been implicated in metastasis [10 ]. Nevertheless, how this chemokine functions in vivo is poorly understood.

Although human CXCL8 (hCXCL8) binds to hCXCR1 and hCXCR2 with comparable affinity, and these two receptors are coexpressed in many cells [17 18 19 ], the differential ligand selectivity of these two receptors suggests that they may function differently [20 ]. Indeed, recent studies have shown that these receptors respond to hCXCL8 in a differential manner and that this may contribute to the varied functions of this chemokine [21 ]. A recent finding of hCXCR1 expression on human CD8+ effector T cells upon antigen stimulation suggests that the regulation of CD8+ T cell homing is mediated by hCXCR1 alone [22 23 24 ]. However, the different roles of these two receptors in hCXCL8 function in humans remain elusive.

Therefore, we took advantage of the murine system to decipher hCXCL8 functions, by developing a conditional, transgenic mouse line containing the hCXCR1 receptor, which can be activated independently in a tissue-specific manner. For these studies, we have used a versatile loxP-based, conditional, transgenic strategy, which involves making a founder mouse carrying a ubiquitous promoter, driving the hCXCR1 transgene with a floxed transcription stop sequence, inserted between the promoter and hCXCR1. This renders the expression of CXCR1 silent in the founder mice until appropriate Cre recombinase is provided to delete the polyA region, thereby activating the reading frame. This setup allows us to achieve tissue-specific targeting of hCXCR1 expression easily by crossing the founder mice with various tissue-specific, Cre recombinase-expressing mice. We have also introduced other useful selection and tracer markers in the construct to increase versatility of this tool to facilitate diverse uses in vitro and in vivo.


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MATERIALS AND METHODS
 
Materials
Plasmid pCAGGS was purchased from Gent University (Belgium), monomeric red fluorescent protein 1 (mRFP1) construct was a gift from Dr. Roger Y. Tsien [University of California, San Diego (UCSD), CA, USA], loxP-enhanced GFP (eGFP)-loxP and pCMV-Cre constructs were gifts from Dr. Frances M. Sladek [University of California, Riverside (UCR), CA, USA], US9-eGFP construct was a gift from Dr. Andrew J. Beavis (Princeton University, Princeton, NJ, USA), and the bicistronic retroviral vector pIB2 was a gift from Dr. Qijing Li (Stanford University, Stanford, CA, USA). hCXCR1-specific antibody was a gift from Genentech (San Francisco, CA, USA). Phospho-ERK antibody was purchased from Cell Signaling Technology (Beverly, MA, USA), total ERK antibody from Upstate Biotechnology (Lake Placid, NY, USA), and GFP antibody was from Invitrogen (Carlsbad, CA, USA). All of the secondary antibodies conjugated to fluorophore were purchased from Molecular Probes (Eugene, OR, USA). PD98059 and Go6983 inhibitors were purchased from Calbiochem (San Diego, CA, USA) and reconstituted in DMSO. All other reagents were from Sigma Chemical Co. (St. Louis, MO, USA), unless mentioned otherwise.

Cell culture
NIH3T3 and HEK293T cells were maintained in DMEM supplemented with 5% of FBS, 1% of penicillin, and 1% streptomycin at 37°C and 5% CO2. Dermal mouse adult fibroblasts and dermal mouse embryonic fibroblasts (MEFs) were isolated from the transgenic mouse founder line as described previously [25 ] and cultured in DMEM with 10% of FBS.

Animal maintenance, genotyping, phenotyping, and tissue processing
The linearized plasmid RCLG3A (pRCLG3A) (~11 kb) DNA was supplied to the University of California, Irvine (CA, USA), Transgenic Mice Facility for transgenic mice generation on a C57B/J genetic background. The tail tissue from 3-week-old pups was used for genomic DNA isolation and genotyping by PCR. Genomic DNA (500 ng) and the unfloxed5/3 primer pair (forward: 5-GGATTACCAGGGATTTCAGTCG-3; reverse: 5-CCTTCTTGGCCTTTATGAGGAT-3) were used in a PCR reaction under the following conditions: 94°C, 1 min; 57°C, 45 s; 72°C, 1 min, for 40 cycles. PCR products were resolved on 1.2% agarose gels, and a 1.2-kb-amplified fragment indicated the presence of the transgene. The Cre-recombinase mouse strains, including Gt(ROSA)26Sortm1(cre/Esr1)Nat (ROSA26-Cre; whole-tissue Cre expression, homozygous) and B6.Cg-TgN(Lck-cre)548Jxm (Lck-Cre; thymocyte-specific Cre expression, homozygous), were purchased from Jackson Laboratory (Bar Harbor, ME, USA). All mice were maintained in the UCR vivarium with a 12-h light/dark cycle and according to the guidelines, approved by the UCR Institutional Animal Care and Use Committee.

To test the ability of Cre to mediate the excision of the floxed region in our transgenic construct, MEFs were isolated from newborn hCXCR1(RCLG3A) transgenic mice and then infected with retrovirus expressing Cre recombinase and cultured in vitro. The cells were treated with tamoxifen (10 ug/ml) for 5 days to induce Cre activation, and then the genomic DNA was isolated for PCR analysis. The sequence upstream of the floxed region is extremely GC-rich (~80% GC); therefore, a special method using 5% DMSO in the PCR reaction [26 ] was used to amplify this region with primer pair floxed5/3 (forward: 5-CCATGTTCATGCCTTCTTCT-3; reverse: 5-CCCAGGACCTCATAGCAAA-3; see Fig. 1A ) using the following cycling conditions: 94°C, 1 min; 59°C, 45 s; 72°C, 3 min, for 35 cycles. The 3.4-kb amplicon indicated the intact transgenic construct before the Cre-mediated excision, and the 0.9-kb amplicon indicated the successful excision of the floxed region from the transgene (see Fig. 1 ).


Figure 1
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  		Figure 1. Schematic diagram of the transgenic vector pRCLG3A. (A) A potent, ubiquitous CMV/β-actin promoter in the vector pRCLG3A was used to drive a series of cassettes, including a floxed mRFP (Marker 1) followed by a triple transcription-stopping polyA sequence (3xPolyA) and a downstream Internal Ribosome Entry Site (IRES)-based bicistronic transcript, including open-reading frames of hCXCR1C4 and a multifunctional marker consisting of firefly luciferase fused to eGFP with a transmembrane-localizing domain (Luc-TM-eGFP). Only mRFP will be transcribed and expressed properly from this construct. (B) When Cre-mediated recombination occurs, the floxed mRFP + 3xPolyA is excised, and the downstream, bicistronic transcript is activated. hCXCR1C4 and the multifunctional marker (Marker 2) will be expressed, replacing mRFP in the Cre-activated cells.

Immunoprecipitation, immunoblotting, and gel electrophoresis
To detect hCXCR1C4 in NIH3T3-expressing hCXCR1, an antibody to this receptor and protein G beads were added into cell extracts to immunoprecipitate hCXCR1C4. The precipitated protein was resolved on 12% polyacrylamide gels and detected using specific antibodies and Dura reagents (Pierce, Rockford, IL, USA), as described previously [27 ].

Transwell chemotaxis assay
These assays were performed as described previously by us [27 ]. Briefly, NIH3T3 cells (2x104) in 100 µl medium were seeded on the bottom side of transwell membranes with 8 µm pore size (BD Biosciences, San Jose, CA, USA), with the under side facing up. The cells were allowed to adhere to the membrane for 30 min at room temperature. After cell adhesion, each transwell was inserted into a 24-well plate with the seeded cells facing down into the lower chamber. Medium was added to the upper (100 µl) and the lower chambers (1 ml). CXCL8 (100 ng/ml) was only added to the upper chambers. The cells were incubated at 37°C for 2 h to allow migration; at the end of the incubation, the cells remaining on the under side of the membrane were removed with a cotton swab, followed by fixation and staining of the membranes with 2% toludine blue in 4% paraformaldehyde. The numbers of cells, which migrated onto the upper side of the membrane, were counted in 10 fields at 10x magnification and averaged.

Luciferase activity assay
Cultured cells were extracted, and luciferase activity was measured following the manufacturer’s instructions using a microplate luminescence reader (LUMIstar) and the luciferase assay substrate, luciferin (Promega, Madison, WI, USA). A minimum of triplicates for each experiment was performed, and the data were normalized to protein concentration and expressed as mean luminescence readings with SE. Tissue luciferase was extracted following a protocol reported previously [28 ] and assayed with the method described above. The data were normalized to dry tissue weight after pulverization.

Immunolabeling and confocal microscopy
Cells were fixed in 4% paraformaldehyde for 15 min, rinsed with PBS, and then quenched in PBS containing 0.1 M glycine for 15 min and blocked with 1% BSA. Primary antibodies (eGFP, 1:1000; hCXCR1, 1:500) in 1% BSA/PBS were applied for 2 h at room temperature and washed, and secondary antibodies were applied for 1 h at room temperature; this was followed by staining of the nuclei using 4',6-diamidino-2-phenylindole (DAPI; Sigma Chemical) at room temperature for 10–20 min. After extensive washes, the cells were mounted in Vectashield (Vector Laboratories, Burlington, CA, USA). Fresh mouse tissues were collected, cut into pieces of 3 mm thickness, fixed in ice-cold 4% paraformaldehyde overnight, rinsed in PBS, quenched with 0.1 M glycine in PBS, and blocked with 1% BSA, and embedded for frozen sectioning. Following section preparation, they were incubated with primary antibody incubation overnight at 4°C. Secondary antibodies were used as described above. Immunofluorescence was imaged using a Leica SP2 scanning confocal microscope. For all the confocal imaging, the machine default capture wavelengths were used, and the entire field was controlled strictly within the capturing dynamic range; therefore, the fluorescent intensity over the entire field shown reflects the real contrast of the tissue specimen without exaggeration or under-represention of the actual fluorescent intensity. All imagines were collected with Z-stacking at small, pinhole settings (0.5–1 air unit), and the raw data were only processed by background subtraction once with the default setting (93.5 µm filter for 40x objective; 35 µm filter for 100x objective). The maximum intensity projection image reconstruction was performed with Imaris software (Bitplane AG, Switzerland), and subsequent, full-size images are shown unaltered at a resolution of 1024 x 1024 pixels.


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RESULTS
 
Generation of the transgenic system
To obtain a hCXCR1 transgenic mouse, which is flexible for the study of the varied biological functions of CXCL8, we designed a construct that brings versatility in the generation of the transgenic mouse in screening and in imaging in vivo and in cells taken from the animal and placed in culture. Based on this concept, we built a vector with a potent and ubiquitous promoter consisting of the CMV enhancer/chicken β-actin promoter and its first intron, and markers, which enable us to monitor the transgene-positive tissues with fluorescence (Markers 1 and 2) and bioluminescence (Marker 2; Fig. 1A ). We chose to use the mRFP1 [29 ] for Marker 1, and for Marker 2, firefly luciferase fused to a membrane-localizing eGFP [30 ]. In Marker 2, eGFP localizes to the extracellular side of the plasma membrane and firefly luciferase to the intracellular domain. The hCXCR1 was modified by fusing this receptor transgene with a tetracysteine motif (C4), which was then introduced into the multicloning site (Fig. 1A) . The C4 motif binds specifically to a group of high-affinity, biarsenical reagents, which allows the fluorescent labeling [31 ] of the recombinant hCXCR1C4 in situ in living cells [32 ] without compromising the function of the protein, as the size of the C4 tag is small. With the appropriate choice of specific, biarsenical reagents, broader use of the C4 motif can be achieved in this transgenic animal, such as the use of electron microscopy [33 ] and chromophore-assisted light inactivation of the protein [34 ]. To activate the expression of hCXCR1, mRFP must be removed by Cre recombinase (Fig. 1B) . We named this vector containing the hCXCR1 transgene RCLG3A.

To test the localization and functionality of hCXCR1, we stably introduced the hCXCR1C4 into murine NIH3T3 cells using retroviruses, enabling us to determine whether the modified hCXCR1C4 is expressed on the cell surface and maintains the functionality of its wild-type counterpart. The infected cells were enriched by antibiotic selection, and hCXCR1C4 expression was confirmed by immunostaining (Fig. 2A ). To test functionality, uninfected and infected NIH3T3 was exposed to hCXCL8 and examined ERK, which is a hallmark for hCXCR1-mediated signaling in human cells [35 ]. When the cells overexpressing the receptor were exposed to hCXCL8, the ERK was phosphorylated/activated, indicating that the receptor is functional (Fig. 2B) . This activation was inhibited partially by a MEK1 inhibitor and considerably inhibited by an inhibitor of PKC, a kinase, which is known to be important in ERK activation [35 ]. This signaling pattern resembles that seen in human cells [35 ]. We also found that when the cells are preincubated with the biaresenical reagent Lumio-Red, this red fluorescent dye binds to the tetracysteine motif in the recombinant hCXCR1C4 protein [31 ]. When the labeled, whole-cell extracts were resolved on a PAGE gel, a fluorescent band with the right molecular weight (~45 kD) was detected with a Texas Red equivalent filter (Fig. 2C) on an Amersham Typhoon 9410 fluorescence gel/blot imager with green excitation laser. This result suggests that the tetracysteine motif is functional in the recombinant hCXCR1C4. To demonstrate further that the recombinant hCXCR1C4 itself is functional, transwell chemotaxis experiments were performed to determine whether hCXCL8 stimulates chemotaxis in cells expressing the C4-tagged hCXCR1 (Fig. 2D) . The cells were plated on the under side of the transwell filter, and the hCXCL8 was placed in the upper chamber. We found that this ligand increased cell chemotaxis (Fig. 2D) . Collectively, these results indicate that the recombinant hCXCR1C4 is a functional substitute of wild-type hCXCR1.


Figure 2
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  		Figure 2. NIH3T3 cells expressing hCXCR1C4. (A) Cell surface hCXCR1C4 localization was demonstrated using immunostaining. (B) Wild-type (WT) and NIH3T3 cells expressing hCXCRC4 were treated with CXCL8 (100 ng/ml) for 10 min in the presence of MEK1/2 (PD98, 3 mM) or PKC (Go6983, 500 nM) inhibitors, and ERK activation was determined by immunoblot using a phospho-ERK (pERK) antibody. (C) Whole cell extracts containing hCXCR1C4 were incubated with Lumio-Red (Invitrogen) to label the C4 motif and then resolved by SDS-PAGE. A fluorescent band was detected with a molecular weight ~45 kD. (D) Wild-type and NIH3T3 cells expressing hCXCRC4 were used in CXCL8-induced transwell chemotaxis assays. After 2 h of CXCL8 stimulation (100 g/ml), cells that migrated across the transwell membrane were counted, and data from the CXCL8-treated group were normalized to that of the untreated group. Cells infected with hCXCR1C4 migrated following CXCL8 treatment, whereas control cells were unable to do so.

Cre-mediated hCXCR1C4 expression and color-switching in vitro
The pRCLG3A plasmid was linearized to produce transgenic mice in a C57B/J genetic background. Tail genomic DNA was isolated and used for genotyping the mice by PCR with the primer pair denoted as "unfloxed5/3" (Fig. 1) . Ten transgenic offspring were identified by this method (data not shown). These founder lines were examined for mRFP expression in their neonatal stage (1 day after birth). Prominent red fluorescence was detected in the neonatal transgenic mice (Fig. 3A ). The mouse line with the highest mRFP expression level was expanded as hemizygotes and used for subsequent experiments.


Figure 3
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  		Figure 3. The RCLG3A transgenic mice were examined for phenotypes and tested for Cre-mediated color-switching and gene expression. (A) Neonatal, transgenic and wild-type littermate mice were examined for full-body expression of mRFP using light microscopy (A1) and fluorescent microscopy (A2). (B) MEFs were isolated from the transgenic neonates and infected with retrovirus expressing Cre recombinase. The expression of Luc/eGFP and hCXCR1C4 as well as the reduced expression of mRFP in these Cre-infected MEFs were determined by immunolabeling of hCXCR1 and fluorescent microscopy to detect mRFP and eGFP. (C) The luciferase activity in MEFs was determined before and after infection with a retrovirus-containing Cre. (D) The Cre-mediated stimulation of hCXCR1C4 and Luc/eGFP expression was determined by immunoblotting. The recombinant Luc/eGFP, a fusion protein of eGFP with firefly luciferase, appears as the combined molecular weight of 100 kD, compared with the wild-type eGFP with a molecular weight of ~30 kD.

Dermal MEFs were isolated from neonatal, transgenic mice and used to determine the ability of Cre to mediate DNA recombination, leading to color-switching in vitro. Transgenic MEFs were infected with retrovirus expressing Cre recombinase for 5 days and then examined for the disappearance of mRFP as shown by a lack of red fluorescence, the activation of eGFP as shown by the presence of green fluorescence, and the expression of hCXCR1C4 by immunolabeling with a specific antibody to this receptor (Fig. 3B) . We also tested luciferase activity and found that the activity of this enzyme is very high in the Cre-activated cells (Fig. 3C) . Furthermore, we used immunoblot analysis to show that hCXCRC4, Luc/eGFP, and eGFP are all highly expressed in the Cre-activated cells when compared with the control cells (Fig. 3D) . These results indicated that in this system, Cre recombinase can mediate hCXCR1C4 expression and color-switching in vitro.

Cre-mediated hCXCR1C4 expression and color-switching in vivo
The RCLG3A transgenic founder mouse line (henceforth designated TG+) has a ubiquitous CMV/β-actin promoter, thus making it possible to activate hCXCR1C4 expression and color-switching with Cre recombinase in any tissue, provided that Cre can be delivered effectively in that tissue. In addition to tissue specificity of hCXCR1C4 expression through tissue-specific Cre, temporal control can be added based on chimeras of Cre, which are inducible upon the administration of specific drugs, such as doxycycline [36 ] or hormone analogs [37 , 38 ]. To prove such principles, we crossed the TG+ with a homozygous line of tamoxifen-inducible Cre driven by the ROSA26 loci [39 ], which delivers moderate levels of Cre-Esr expression in most tissues in mice (henceforth designated as ROSA26Cre-Esr+/–). Esr is a chimeric protein of Cre fused to a mutant estrogen-binding domain, which has high affinity to the estrogen synthetic analog tamoxifen. The recombinant Cre-Esr protein is normally sequestered in the cytoplasm, but upon tamoxifen treatment, it translocates into the nucleus, where Cre can make contact with recombine chromosomal DNA. Eight-week-old offspring from this crossing (TG+/ROSA26Cre-Esr+/–) were treated with tamoxifen via intraperitoneal injection for 5–8 days. At the end of the treatment period, we profiled organs for mRFP -> eGFP color-switching and for tissue hCXCR1C4 expression.

At the body-part level, after 5 days of tamoxifen treatment, mRFP -> eGFP color-switching was obvious in the paws (Fig. 4A ) and in organs such as the kidney (Fig. 4B) ; the mRFP levels were reduced significantly by 8 days of treatment. The apparent low-level fluorescence depicted in the organs of the wild-type animals is a result of the fact that collagen-containing tissues frequently display autofluorescence, especially in the green channel [40 ]. This is also evident below (see Go Go Go Go Fig. 9B ). However, this autofluorescence is much weaker than any of the true eGFP signals.


Figure 4
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  		Figure 4. Crossing of the TG+ with ROSA26Cre-Esr+/– to visualize color-switching in body parts. Littermates [wild-type (TG–/ROSA26Cre-Esr+/–) or double-positive offspring (TG+/ROSA26Cre-Esr+/–)] were examined for Cre-induced mRFP to eGFP fluorescent color-switching in paws (A) and kidneys (B). When Cre activity was induced in the animals for 5 days by tamoxifen treatment (Tam; 2 mg/day), eGFP was expressed. By Day 8 of tamoxifen induction, the mRFP has been reduced significantly and replaced by eGFP. In wild-type mice, low levels of autofluorescence were detectable in the green fluorescence channel.


Figure 5
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  		Figure 5. Crossing of the TG+ with ROSA26Cre/Esr+/– to visualize color-switching and CXCR1 expression in tissues. The TG+ was crossed with ROSA26Cre-Esr+/–, and subsequent double-positive offspring (TG+/ROSA26Cre-Esr+/–) were fed with tamoxifen (1.5 mg/day) to induce Cre activity. The tamoxifen-induced mRFP-to-eGFP fluorescent color-switching and expression of hCXCR1 were subsequently examined in mice before (Columns 1–3) and after (Columns 4–6) tamoxifen administration. Paraformaldehyde-fixed, frozen tissue sections of (A) heart, (B) liver, and (C) skeletal muscle were examined with confocal fluorescent microscopy with four channels, including mRFP (red channel), eGFP (green channel), hCXCR1 (magenta channel), and nuclei (blue channel). *, For orientation, some structures are indicated.


Figure 6
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  		Figure 6. Crossing of the TG+ with ROSA26Cre-Esr+/– to visualize color-switching and CXCR1 expression in other tissues. The TG+ was crossed with ROSA26Cre-Esr+/–, and subsequent double-positive offspring (TG+/ROSA26Cre-Esr+/–) were fed with tamoxifen (1.5 mg/day) to induce Cre activity. The Cre-induced expression of hCXCR1 was subsequently examined in paraformaldehyde-fixed, frozen tissue sections of (A) kidney, (B) lung, and (C) skin.


Figure 7
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  		Figure 7. Crossing of the TG+ with Lck-Cre+/+ to visualize color-switching and CXCR1 expression in thymocytes and T cells. The TG+ was crossed with Lck-Cre+/+, and subsequent double-positive offspring (TG+/Lck-Cre+/+) was examined for expression of hCXCR1 in thymocytes and T cell-enriched tissues: (A) spleen, (B) lymph node, and (C) thymus.


Figure 8
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  		Figure 8. Crossing of the TG+ founder line with ROSA26Cre-Esr+/– to visualize luciferase activity. The TG+ founder line was crossed with ROSA26Cre-Esr+/–, and subsequent double-positive offspring (TG+/ROSA26Cre-Esr+) was fed tamoxifen (1.5 mg/day) to induce Cre activity. (A) Cre-induced luciferase activities were examined in mouse tissues. Luciferase activities were normalized to tissue weight, and an arbitrary average zone (red line) was identified based on the luciferase activities in a majority of tissues. (B) Noninvasive imaging of bioluminescence emitted from mice of different genotypes was performed. Mice were injected with luciferin (4 mg) via intraperitoneal injection, and bioluminescence was measured with a sensitive charged-coulpled device (CCD) camera. Exposure times are indicated in each figure. Several organs, including the liver (arrow) and pancreas (arrowhead) were found to have prominent luciferase activity.


Figure 9
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  		Figure 9. Functional test of hCXCR1 in transgenic mice. (A) MEFs from TG+/ROSA26-Cre-Esr+/– were cultured, treated with tamoxifen to activate Cre recombinase and expression of hCXCR1, and then treated with IL-8. ERK phosphorylation was examined to determine hCXCR1-mediated signal transduction. (B) TG+/Lck-Cre+/+ mice, which express hCXCR1 in T cells, and TG+ mice, which contain but do not express hCXCR1, were subject to skin excisional wounds followed by 5 days of topical IL-8 treatment. IL-8/hCXCR1-mediated influx of T cells toward the wound tissue was observed (indicated by the green T cells with arrowheads in wound tissue). In the mice, in which T cells express hCXCR1 (TG+/Lck-Cre+/+), these leukocytes were attracted by IL-8 to the wound tissue, whereas those which do not express hCXCR1 (TG+) T cells were not seen in the wound tissue.

At the tissue level, tissue sections were examined with laser-scanning confocal microscopy for the mRFP -> eGFP switch. Five days of tamoxifen treatment resulted in a decrease of mRFP and an increase of eGFP expression in most tissues; however, the degree of color-switching varied. Here, we illustrate this finding in three tissues: cardiac muscle (Fig. 5A , 1–6), liver (Fig. 5B , 1–6), and skeletal muscle (Fig. 5C , 1–6). In all cases, as the red from mRFP disappears (Fig. 5 , Column 4), the green from eGFP appears (Fig. 5 , Column 5). Simultaneously, we see that hCXCR1 is expressed (Fig. 5 , Column 6), and the receptor is localized to the periphery of the cells. These findings indicate that the Cre recombinase has removed the mRFP and activated the biscistronic gene complex hCXCR1/Luc/eGFP. Figure 6 illustrates other tissues stained for hCXCR1 in the TG+/ROSA26Cre-Esr+/– transgenic mice. In the kidney, this receptor is found in the cells surrounding glomeruli, presumably the cells of the Bowman’s capsule, and in the cells lining the glomerular capillaries. In some cases, large cells are also staining inside the glomeruli themselves; these may be mesangial cells. In the lung, hCXCR1 is expressed in the alveoli and alveolar bronchi, and in the skin, this receptor is found primarily in the mesodermal tissue.

In addition to examining the expression of hCXCR1 using the ROSA26Cre-Esr+/– mouse, we crossed TG+ mice with an Lck promoter-driven Cre (Lck-Cre+/+), which has thymocytes, and mature T cells restricted Cre activity [41 ]. In the thymus, lymph nodes, and spleen of adult mice from this cross, we detected numerous hCXCR1-expressing cells (Fig. 7 ).

As mentioned previously, in the TG+/ROSA26Cre-Esr+/– mice, tamoxifen stimulation of Cre activity activates the expression of Luc, which is a functional firefly luciferase, enabling highly quantitative and reproducible, noninvasive imaging of bioluminescence in live animals [42 ]. In all of the tissues examined, the pancreas has the highest relative luciferase activity (photon yield/tissue weight), which is one to two orders of magnitude above the luciferase activity of most organs (Fig. 8A ). Liver and skeletal muscle also have high luciferase activity, approximately one order of magnitude above average levels. Therefore, when tamoxifen was fed to these TG+/ROSA26Cre-Esr+/– mice, considerable luciferase activity was detected in various organs. These results correlate well with those presented with tissue staining for hCXCR1 and eGFP. For example, brain and small intestine show low luciferase activity (Fig. 8A) , which also correlates well with the fact that these tissues did not show much eGFP or hCXCR1 staining (data not shown). To evaluate whether the luciferase activity in these mice is sufficient for bioluminescence imaging in the whole animal, we anesthetized the mice and injected luciferin, the substrate for luciferase, via intraperitoneal injection, and then measured the bioluminescence emitted from the ventral side of these animals using a sensitive CCD camera. Bioluminescence could be detected locally in the body of the mouse with 5 min exposure (Fig. 8 , B1), and by 15 min, we observed saturation in most parts of the body (Fig. 8 , B2), suggesting that the luciferase activity levels in these mice are sufficient to carry out in vivo bioluminescent imaging. Several areas showed intense luminescence, such as liver, pancreas, and muscle, and these observations correlate well with the luciferase activity tissue profiling (Fig. 8B) . The face, paws, genital area, and tail also have high luciferase levels.

Function analysis of hCXCR1 in the transgenic mice
To determine whether hCXCR1 is functional in vivo, we took advantage of the fact that IL-8 activation of CXCR1 and CXCR2 can rapidly stimulate ERK activation/phosphorylation. MEFs were isolated from TG+/ROSA26Cre-Esr+/– mice. In the absence of tamoxifen treatment, these MEFs only responded weakly to IL-8 stimulation, presumably as a result of the endogenous mouse (m)CXCR2 (Fig. 9A ). However, after tamoxifen treatment, IL-8 stimulated significant ERK activation/phosphorylation (Fig. 9A) , indicating that the hCXCR1 expressed in vivo is functional.

In addition to the experiments described above to demonstrate functionality of CXCR1 in our mouse system, we performed experiments to further the recent finding that in humans, hCXCR1 is found in T cells [23 ]. For these experiments, we used the TG+/Lck-Cre+/+ mice, which express the hCXCR1 gene specifically in T cells, to test whether in these mice, IL-8 attracts the labeled T cells to sites of injury. Excisional wounds were performed on the back of these mice, and IL-8 (500 ng) was applied topically to the wound bed for 5 consecutive days, followed by collection of the wounded tissue to analyze for the presence of green lymphocytes. Day 5 was chosen to allow for the infiltration of lymphocytes, as the first leukocytes to come to the wound site are neutrophils, closely followed by macrophages, and a few days later, by lymphocytes. We found that in control mice, no T cells were detected in the skin wound tissue (Fig. 9B) . However, the IL-8 treatment resulted in many T cells being chemoattracted to the wound site of the TG+/Lck-Cre+/+ mice, suggesting a novel function for IL-8/CXCR1, which is that IL-8 is able to chemoattract specific, mature T cells.


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DISCUSSION
 
hCXCL8 has two receptors, hCXCR1 and hCXCR2, but the latter binds a variety of other chemokines, whereas the former binds hCXCL8 specifically. These receptors are coexpressed in many human cells, and they are both functional, although the contribution of each to particular hCXCL8-mediated effects is not clear. However, more recently, it has been shown that only hCXCR1 is expressed in human CD8+ effector T cells [21 ] upon antigen stimulation, suggesting that CXCR1, but not CXCR2, contributes to regulation of CD8+ T cell homing [22 23 24 ]. In the past few years, the existence and function of mCXCR1 have been in dispute. More recent studies have suggested the existence of a mouse homologue of hCXCR1 [43 ] and that this potential "mCXCR1" is a functional receptor for mouse granulocyte chemotactic protein-2 (GCP-2) in vivo [44 ]. The mouse homologue of CXCL8 has not been found, even after careful examination of the whole mouse genome (National Center for Biotechnology Information, Mouse Genome Resources, http://www.ncbi.nlm.nih.gov/genome/guide/mouse/). The humanized mouse system we present here will be useful in investigating multiple functions of hCXCL8 in vivo.

A considerable amount of effort has been used to optimize transgenic mouse techniques in the past decade, and numerous strategies have been developed to achieve highly controlled gene expression in mice [45 ]. Most of these strategies are derived from the Cre/loxP recombination system [46 ] or the tetracycline-inducible system [47 ]. The core of the Cre/loxP system relies on the use of P1 phage Cre recombinase and its derivatives to catalyze the excision of the DNA sequence flanked by loxP sites on the chromosome [48 ]. Use of this system usually involves two mouse lines, one of which carries the transgene with a floxed transcription "stop sequence" inserted between the promoter and the transgene to silence transgene expression and another mouse line expressing Cre recombinase [46 ]. Tissue-specific expression of the transgene can be achieved by choosing the mouse line expressing Cre under the appropriate tissue-specific promoter to cross with the transgenic line. In addition to regulating transgene expression in a tissue-specific manner, temporal control of transgene expression can be achieved to circumvent embryonic lethal phenotypes. This is usually done by linking the Cre gene with a drug-inducible promoter [49 ] or generating a chimeric Cre protein, which is normally sequestered in the cytoplasm, but whose nuclear translocation is inducible by specific, synthetic steroids [50 ].

The transgenic system we developed here offers: Cre/loxP-mediated, spatially and temporally controlled hCXCR1 expression; a color-switching mechanism, which uses spectrum-complementary, genetically encoded green and red fluorescence markers to label the hCXCR1-expressing cells (eGFP+) against the background (mRFP+); a bioluminescent marker, which is expressed in the hCXCR1-expressing cells; an exogenous cell surface marker (eGFP moiety) in the hCXCR1-expressing cells, which facilitates the isolation or targeting of these cells. As a result, the established transgenic founder line RCLG3A only expressed mRFP and did so in a ubiquitous manner. When RCLG3A was crossed with the tamoxifen-inducible, whole-tissue Cre mice (ROSA26-Cre/Esr), administering tamoxifen induced whole-body hCXCR1 expression and color-switching. When RCLG3A was crossed with thymocyte-specific Cre mice (Lck-Cre), the hCXCR1 expression and color-switching were restricted in a lineage-specific manner. This mouse line is useful in establishing tissue-specific, transgenic mice in conjunction with imaging-ready, multifunctional cell markers, which facilitate cell imaging, tracing, and manipulations in vivo.

mRFP has been developed recently as an alternative to the tetrameric red fluorescence protein, dsRed, originally identified in coral reef Discosoma species. dsRed had several disadvantages, which prevented its prevalence in cell biology. The maturation of dsRed protein is slow, and the mature protein is an obligated tetramer with low solubility. mRFP1, unlike its parent dsRed, is biocompatible with mammalian cells, as its mature form is a monomer instead of an insoluble tetramer. In addition, it has a 20-nM emission red shift from dsRed, which gives it an ideal complementary emission spectrum to eGFP [29 ]. Therefore, in our construct, we used the mRFP as an animal-screening tool and for counter-staining [51 ]. The mRFP-expressing cells can be distinguished from eGFP-expressing cells with minimal emission overlap. In the case of crossing with the ROSA26 loci, the partial mRFP retention in some tissues can be explained by the low-to-moderate Cre activity delivered by this Cre line, which is decreased further and limited by factors such as Cre heterozygosity and tamoxifen penetration and potency.

In our system, hCXCR1 has been modified with an extra 6-amino acid tetracysteine motif (C4) on the C terminus of the wild-type protein (Fig. 1) . The C4 motif allows fluorescent labeling [31 ] of the recombinant hCXCR1C4 in situ and in live cells [32 ] without compromising the function of the receptor, as the tag is small. With appropriate use of the biarsenical reagents, broader use of the motif can be coupled to this transgenic animal, such as electron microscopy [33 ] and chromophore-assisted light inactivation of the protein [34 ], through the use of different types of biarsentical labeling reagents. Biarsentical labeling reagents are not fluorescent by themselves, but upon binding to the C4 tag, they become fluorescent and thus, can be used to label the recombinant proteins in live cells in real time. The C4 tag did not alter the membrane localization of hCXCR1. This receptor was detected predominantly on the cell surfaces in solid organs, skeletal muscle, and leukocytes. The successful stimulation of hCXCR1C4 expression by Cre in most tissues suggests parallel color-switching events in these tissues.

We did not test the C4 reagents in vivo, as these reagents are relatively novel, and although their usefulness has been shown in cultured cells, it has not been shown in animals. There are two reasons that this technique might not be suitable for live animals. One is that these reagents (Lumio-Red and Lumio-Green) contain arsenic, and their degraded by-products might be toxic to the body. The other reason is that the staining process of the Lumio dyes requires the presence of µM levels of 1, 2-ethanedithiol (stabilizer and solubilizer), which does not kill the cells in culture but might be deleterious to the animal. Therefore, we added this marker primarily for study of cell function when the cells have been removed from the animal and cultured.

The fusion marker Luc-TM-eGFP plays important roles in our system after Cre activation. However, prior to Cre-mediated recombination, the expression of this marker must be suppressed fully; thus, a triplet polyA signal was inserted after mRFP (Fig. 1) , which prevents transcriptional read-through of a single polyA sequence efficiently. The Luc/eGFP is present on the same bicistronic transcript with hCXCR1C4, but its translation is driven by an IRES [52 ], hence its expression level parallels that of hCXCR1C4. Luc/eGFP was designed to bring together numerous functions. It consists of a functional eGFP on its C terminus and a functional firefly luciferase on its N terminus, thereby generating fluorescence and bioluminescence in cells. This fusion protein localizes to the cell surface; the extracellular eGFP moiety serves as an exogenous motif, which can be used for antibody-mediated cell isolation by MACS, and the luciferase activity in the intracellular domain can also be targeted for in vivo imaging.

We are still characterizing the phenotype of the mice after Cre induction and plan to perform further experiments to study the function of hCXCR1 in the mice with human IL-8 application. The mice homologue for human IL-8 is not present in the genome or is still undiscovered; in mice, the only known ligand for hCXCR1 is mGCP-2. It is possible that hCXCR1 expression alone in mice has a spontaneous phenotype without being activated by IL-8, but if so, this phenotype is probably subtle and will require considerable investigation to reach conclusions. Thus far, the mice do not appear to have inflammatory disorders.

In summary, we have established a transgenic mouse line for expression of hCXCR1, which incorporates the Cre/loxP system into a dual-fluorescent and bioluminescent color-switching system and delivers controllable expression of the hCXCR1 gene in a spatial or temporal manner. This mouse line is amenable to multiple types of fluorescent and bioluminescent imaging for hCXCR1-positive tissues/cells and brings great simplicity for mice phenotyping, primary cell isolation, and targeting in vivo with the implementation of multifunctional cellular markers.


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ACKNOWLEDGEMENTS
 
This research was supported in part by National Institutes of Health R03 grant 5R03AI068139-02 to M. M-G. We thank Melissa Petreaca of the Department of Cell Biology and Neurosciences at UCR for helpful discussions and comments about the manuscript. We are indebted to Dr. Frances Sladek (UCR), Dr. Qijing Li (Stanford University), and Dr. Roger Tsien (UCSD) for providing specific plasmid DNA constructs.

Received March 8, 2007; revised May 30, 2007; accepted June 8, 2007.


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