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发表于 2009-3-5 00:02 |只看该作者 |倒序浏览 |打印

作者：Gerald J. Spangrudea,b, Scott Chob, Otto Guedelhoeferc, Ryan C. VanWoerkoma, William H. Flemingd 6 M! L3 H% G' _3 L6 g( a6 f$ F
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      【摘要】
   Transgenic mouse strains ubiquitously expressing green fluorescent protein (GFP) have enabled investigators to develop in vivo transplant models that can detect donor contributions to many different tissues. However, most GFP transgenics lack expression of the reporter in the erythroid lineage. We evaluated expression of GFP in the bone marrow of the OsbY01 transgenic mouse (B6-GFP) in the context of CD71 and TER-119 expression and found that GFP fluorescence is lost prior to the basophilic erythroblast stage of development. However, platelets in B6-GFP mice were found to be uniformly positive for GFP. We therefore used the GFP transgenic model in combination with allelic variants of CD45 and the hemoglobin &szlig; (Hbb) chain to develop a model system that allows all blood lineages to be followed in a mouse model of bone marrow transplantation (BMT). To detect Hbb variant molecules, we developed a new protocol based on high-performance liquid chromatography that is sensitive and precise, allowing rapid and quantitative analysis of erythroid chimerism. Platelet and leukocyte engraftment were detected by flow cytometry. BMT into sublethally irradiated (4 Gy) recipients demonstrated the failure of B6-GFP-derived cells to engraft relative to B6-CD45a-derived cells, suggesting that an immune barrier may prevent efficient engraftment of the transgenic cells in a setting of minimal ablation. These results establish limitations in the use of transgenic GFP expression as a donor marker in transplantation models.
      【关键词】 Erythropoiesis Hematopoietic chimerism Hematopoietic cell transplantation Engraftment' ~& G. z9 g5 c
   INTRODUCTION
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A number of mouse transplant models are available for analysis of engraftment following bone marrow transplantation. Traditionally, erythroid engraftment has been followed using naturally occurring variants of the hemoglobin ß (Hbb) chain that have been bred onto a common genetic background , severely limiting simple analysis of erythroid engraftment by this method. Here, we combine CD45 and Hbb alleleic strains with the transgenic GFP model to compare relative efficiencies in detecting engraftment using the different donor markers. To facilitate analysis of erythroid engraftment, we developed a new high-performance liquid chromatography (HPLC) method capable of resolving the Hbbd and Hbbs variants.8 A- [% Q, ^( x
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MATERIALS AND METHODS2 I) H3 Q2 _/ q

Animals

Mice carrying the Thy-1.1 and Ly-5.1 alleles on a C57BL background were generated in our breeding colony by mating the BKa.AK-Thy1a/Ka and B6.SJL-Ptprca Pep3b/BoyJ strains and selecting for cosegregation of Thy-1.1 (CD90a) and Ly-5.1 (CD45a). Hereafter, we refer to these mice as B6-CD45a. Breeding pairs of B6.Cg-Gpi1a Hbbd H1b/DehJ mice  and are here referred to as B6-GFP. All animals were maintained in the animal resources center at the University of Utah under protocols approved by the institutional animal care and use committee. All mice were maintained on sterilized food and acidified water to prevent colonization by Pseudomonas.3 T8 r6 g& m7 x) M

Flow Cytometry
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Multiparameter flow cytometric analysis was performed using FACScan and FACSVantage instruments (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com). Bone marrow samples were prepared by flushing isolated femurs with Hanks¡¯ balanced salt solution containing 5% newborn calf serum (HyClone, Logan, UT, http://www.hyclone.com). Peripheral blood samples were collected from the retroorbital sinus under isoflurane anesthesia (IsoSol; Vedco Inc., St. Joseph, MO, http://www.vedco.com) delivered using the E-Z Anesthesia system (Euthanex Corp., Palmer, PA, http://www.ezanesthesia.com/index2.htm). Blood was collected in heparinized capillary tubes and mixed with acid citrate dextrose at a 10:1 ratio prior to determination of the complete blood count using a Serono System 9010 CP hematology counter (Serono Diagnostics, Allentown, PA). Samples were then mixed with an equal volume of 2% Dextran T500 (Amersham Biosciences, Piscataway, NJ, http://www.amershambiosciences.com) in phosphate-buffered saline (PBS) and incubated at 37¡ãC for 30 minutes. The upper layer, containing leukocytes, platelets, and residual erythrocytes, was collected for cytometric analysis, whereas the sedimented erythrocytes were washed twice in PBS and stored as a packed cell pellet at ¨C20¡ãC prior to Hbb analysis.
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Cell surface antigens were identified using the following antibody conjugates: allophycocyanin-conjugated Ly-5.1; phycoerythrin-conjugated CD4, CD8, CD19, CD117, and Ly-6G (eBiosciences, San Diego, CA, http://www.ebioscience.com); phycoerythrin-conjugated CD11b and biotin-conjugated CD71 (BD Biosciences). TER-119 was purified from conditioned medium in our laboratory and detected using a phycoerythrin-conjugated anti-rat  light chain reagent (BD Biosciences), followed by blocking with 10 µg of an isotype-matched monoclonal antibody (clone 53-7.3, anti-CD5). Streptavidin-conjugated Alexa Fluor-647-R-phycoerythrin was purchased from Invitrogen (Carlsbad, CA, http://www.invitrogen.com). Dead cells were excluded from analysis using forward and side scatter and propidium iodide gating.8 G& p! s) M8 T# x0 q3 x
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Determination of Hbb Variants by Electrophoresis  U) m! y) c! P7 e0 `

Packed erythrocyte samples were lysed by freeze-thaw and clarified by centrifugation at 12,000g for 2 minutes. Cystamine derivatization was performed by mixing 20 µl of clarified erythrocyte lysate with 150 µl of a solution containing 67 mM cystamine dihydrochloride, 1.2 mM dithiothreitol, and 100 mM (0.2% wt/vol) ammonium hydroxide ) and quantitated using ImageJ software, version 1.33u (NIH, Bethesda, MD, http://rsb.info.nih.gov/ij).
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Determination of Hbb Variants by HPLC
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A cation exchange protocol was developed to separate and quantitate Hbb variant alleles by HPLC. A stock solution of 100 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) was prepared by dissolving 10 mg of DTNB in 250 µl of dimethyl sulfoxide and stored at ¨C20¡ãC. Erythrocytes obtained from heparinized blood were washed three times in dextrose-gelatin-veronal buffer and stored as packed pellets at 4¡ãC for up to 2 weeks prior to analysis. Samples were derivatized by adding 5 µl of packed erythrocytes to 100 µl of 40 mM NaCl, 1 mM DTNB and incubating at room temperature for 30 minutes. Following centrifugation (12,000g for 2 minutes), samples (20 µl) were injected onto a strong cation exchange column (Shodex IEC SP-825; Phenomenex, Torrance, CA, http://www.phenomenex.com) equilibrated in Buffer A (10 mM citrate, 12.5 mM NaCl, 25 µM thimerosal, pH 5.88), using a Beckman System Gold model 125 solvent module. A convex gradient to Buffer B (10 mM citrate, 30 mM NaCl, 25 µM thimerosal, pH 6.0) was applied over 3.5 minutes at a flow rate of 2 ml/minute and held for 2.9 minutes, followed by a high-salt wash (350 mM NaCl). The column was re-equilibrated in Buffer A between samples for 3 minutes at 2 ml/minute. Protein peaks were detected at 280 and 415 nm after subtraction of background detected at 490 nm, using a Beckman System Gold model 168 diode array detector module. The Hbbd variant eluted at 2.9 ¡À 0.2 minutes, whereas the Hbbs variant eluted at 5.6 ¡À 0.1 minutes (mean ¡À SD; n = 8). Absorbance data collected at 415 nm were integrated using Beckman System Gold software version 8.1.0 to determine percentage of Hbbs and Hbbd in experimental samples.

Bone Marrow Transplantation
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To directly compare the GFP and CD45 systems in a transplant model, bone marrow cells obtained from B6-CD45a and B6-GFP donor animals were mixed at a 50:50 ratio. Radiation was delivered to B6-Hbbd recipient mice in a single dose (4 Gy) or in a split dose (2 x 4 or 2 x 6 Gy) with a 3-hour interval between doses, using a Shepherd Mark I 137Cs source (JL Shepherd and Associates, Glendale, CA, http://www.jlshepherd.com) at a dose rate of 0.8 Gy/minute. Bone marrow cells were transplanted by the retro-orbital route under isoflurane anesthesia at a dose of 2 x 106 total cells in 0.2 ml. Peripheral blood samples were collected weekly for analysis of trilineage donor-derived engraftment, as described above and shown in Figure 1.

Figure 1. Identification and phenotyping of peripheral blood leukocytes, platelets, and erythrocytes by flow cytometry. Peripheral blood was collected from a supralethally irradiated (2 x 6 Gy) B6-hemoglobin ß (Hbb)d recipient 4 weeks after transplantation of bone marrow cells obtained from B6-CD45a and B6-GFP donor animals (1 x 106 from each donor). (A): Erythrocyte-depleted peripheral blood was analyzed by forward and side scatter to identify platelets, residual erythrocytes, and leukocytes, as shown by the boxed regions. (B): Analysis of cells falling within the leukocyte gate demonstrating three populations, which are derived from the two donor marrow grafts and from endogenous hematopoietic recovery (GFPnegCD45a-neg). (C): Analysis of cells falling within the platelet gate, demonstrating expression of GFP by 68% of the platelets. (D): Analysis of cells falling within the erythrocyte gate, demonstrating expression of GFP by 2.5% of erythrocytes. Abbreviation: GFP, green fluorescent protein.
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To confirm the engraftment barrier against B6-GFP bone marrow cells in sublethally irradiated B6-Hbbd recipient mice, we transplanted 2 x 106 bone marrow cells obtained from B6-CD45a or B6-GFP donor animals into separate groups of irradiated (4 Gy) recipients. Peripheral blood samples were collected weekly for analysis of trilineage donor-derived engraftment, as described above.

RESULTS) p; Y, Q: m6 ~1 B. n  P

Strategy for Comparison of CD45 and GFP as Markers of Donor Engraftment

To directly compare the efficiency of the CD45 and GFP markers in a transplant setting, we designed an experiment in which bone marrow cells obtained from B6-CD45a and B6-GFP donors were mixed and transplanted into irradiated B6-Hbbd recipient mice (Table 1). If the two donor populations function with equal efficiency at engraftment in the recipient animals, we would predict equal representation of cells expressing each marker in all blood lineages. Because CD45 is not expressed by erythrocytes or platelets, however, we were only able to directly compare engraftment of the two bone marrow samples in the leukocyte lineages. We expected to find GFP expression in erythrocyte and platelet lineages, as well as leukocyte lineages. Figure 1 shows a flow cytometry analysis of a 2 x 6 Gy irradiated recipient evaluated 4 weeks after transplant. We were able to identify platelet, erythroid, and leukocyte subsets of peripheral blood samples based on forward and side scatter characteristics, as shown in Figure 1A. Gating on the leukocyte subset, we evaluated expression of GFP and CD45a and found these markers to be present in equal frequencies on mutually exclusive populations, as expected (Fig. 1B). However, although GFP was expressed on approximately 70% of the platelets in this chimeric mouse (Fig. 1C), consistent with the 1:1 transplant of GFPpos:GFPneg bone marrow cells, very little GFP was detected in the erythrocyte lineage (Fig. 1D). This result indicates that although the particular B6-GFP donor mouse strain used in this study allows leukocyte and platelet chimerism to be measured, donor-derived contributions to the erythroid lineage cannot be distinguished from endogenous hematopoietic recovery.! O4 |' A" h+ D  ~1 O! F6 M0 ^% j

Table 1. Mouse strains and markers used in transplant experiments! p  X. S0 z+ Q' R

Detection of GFP in Blood Cells Obtained from B6-GFP Transgenic Mice
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To determine the stage of erythroid development at which GFP fluorescence is lost, we evaluated the bone marrow of B6-GFP mice with respect to three antigens associated with erythroid differentiation  and largely retains a GFPhigh phenotype (Fig. 2A, R6).

Figure 2. Analysis of GFP expression throughout erythroid lineage development in the bone marrow of B6-GFP mice. (A): Bone marrow cells obtained from a B6-GFP donor were stained with allophycocyanin-CD117 and biotin-CD71 antibodies, followed by streptavidin-phycoerythrin-Alexa-Fluor 647. Dead cells were excluded by propidium iodide and scatter gating. The contour plot represents 80% log density. Erythroid development proceeds from a CD117high CD71neg stage (R1) through intermediate stages (R2¨CR4) to the basophilic erythroblast stage (R5). Lymphoid development proceeds through a CD117low CD71low intermediate (R6). Analysis of GFP expression by the gated populations demonstrates progressive loss of GFP fluorescence during erythroid, but not lymphoid, differentiation. (B): Bone marrow cells obtained from a B6-GFP donor were stained with TER-119 followed by phycoerythrin-anti-rat, blocked with an isotype-matched control, and followed with biotin-anti-CD71 and streptavidin-phycoerythrin-Alexa-Fluor 647. Dead cells were excluded by propidium iodide and scatter gating. The contour plot represents 80% log density. Erythroid development proceeds from the basophilic erythroblast stage (R2) to the orthochromatic erythroblast and later stages (R4). GFP expression is already decreased by 100-fold at the basophilic erythroblast stage compared with earlier progenitors (R1) and is progressively lost during maturation. Abbreviation: GFP, green fluorescent protein.

GFP Fluorescence Is Lost Prior to the Terminal Stages of Erythroid Development

The lack of GFP fluorescence in the erythroid lineage of B6-GFP mice could be due to degradation of GFP protein over the lifespan of the erythrocyte, decreased synthesis during erythroid development, or both. To further clarify expression of the transgene during erythroid development, we evaluated CD71 expression with respect to TER-119. TER-119 is expressed from the proerythroblast stage of erythroid development and persists through enucleation and throughout the life of the erythrocytes. In contrast, CD71 expression is extinguished at the orthochromatic erythroblast stage of development . Thus, transgenic GFP expression is likely silenced at the transcriptional level in the erythroid lineage, depending on the site of integration into the genome.- o+ Q7 s2 D$ h( K2 ^

Detection of Hemoglobin Variants by HPLC

Allelic variants of mouse Hbb can be distinguished by alkaline gel electrophoresis. The Hbbs variant found in C57BL strain mice differs from the Hbbd variant by a Cys  Gly substitution at position 13. Reduction of thiol groups with cystamine introduces a differential charge due to this allelic variation that allows clear resolution of the two alleles by agarose electrophoresis under alkaline conditions . However, the gel electrophoresis detection system is cumbersome for routine analysis and can present problems of interpretation and quantitation due to irregularities in the gel and underloading or overloading of samples. We therefore developed an automated HPLC method for mouse Hbb analysis (Fig. 3A). Mouse erythrocyte lysates are first reacted with DTNB to reduce free thiols with an acidic moiety. Lysates are then separated by cation exchange chromatography under conditions that resolve Hbbs from Hbbd due to the Cys  Gly substitution at position 13 in Hbbs. The acidic charge introduced into Hbbd by the DTNB derivatization reduces the retention time of Hbbd to 2.9 minutes, compared with 5.6 minutes for Hbbs. The two peaks are readily resolved in less than 10 minutes (Fig. 3A). Alkaline gel electrophoresis after reduction with cystamine also resolves the two isoforms (Fig. 3B) but requires quantitation by scanning of the gel. A comparison of artificial mixtures of lysates of the two isoforms showed that although both methods exhibited a linear relationship between measured and actual percent Hbbs (Fig. 3C), the limit of detection for the gel technique was 10%, whereas the HPLC technique detected as little as 1% variant Hbb isoform. Correction of the gel data by background subtraction improved resolution at the extremes of the dose-response curve but also led to a nonlinear relationship between measured and actual percent Hbbs. These data demonstrate that the HPLC method is superior to gel electrophoresis for measuring erythroid chimerism. Since the DTNB-treated lysates are relatively stable, batches of samples can be prepared and run unattended using an HPLC autosampler. Unlike gel electrophoresis, analysis of HPLC data does not require the manual process of quantitation by scanning and integrating the gel bands. Therefore, the HPLC technique represents an automated, rapid throughput approach for routine analysis of mouse Hbb isoforms.. y5 ?" ^; S( V4 Z6 I+ d

Figure 3. Detection of Hbb allelic variants by HPLC. (A): Elution profiles of samples obtained by mixing Hbbs and Hbbd blood in defined ratios. The actual percentage of the B6 allelic variant (Hbbs) is noted above each sample. (B): Control mixtures of the indicated percentages of Hbbs and Hbbd samples were analyzed by alkaline gel electrophoresis. The origin of application () and polarity of the gel ( ) are indicated. The dried gel was photographed and analyzed using ImageJ software to integrate the band densities. (C): Data obtained by HPLC and gel electrophoresis methods were plotted to demonstrate the relationship between actual and measured percent Hbbs as determined by the two techniques. HPLC results are linear (r2 = .998) and can accurately detect 1% Hbbs in a mixture of the two isoforms. To determine the precision of the HPLC method, we evaluated a sample obtained by mixing 5 µl of Hbbs lysate with 20 µl of Hbbd lysate in a series of eight sequential HPLC runs and found that the assay detected 21.5 ¡À 1.8% Hbbs (mean ¡À SD; range = 18.5%¨C24.0%). Raw gel data indicate that the background density in the gel was not subtracted from the peaks, whereas corrected gel data include background subtraction. Raw gel data are linear but have a limit of detection of 10%. Abbreviations: Hbb, hemoglobin ß; HPLC, high-performance liquid chromatography.

Analysis of Trilineage Engraftment Kinetics in a Mouse Model of Bone Marrow Transplantation- J. v2 g1 E- E0 W0 U4 t
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We compared engraftment of B6-CD45a and B6-GFP bone marrow cells in a competitive setting, where equal numbers of cells from each donor strain were mixed and injected into B6-Hbbd recipient mice (Fig. 4). Recipients were conditioned with sublethal (4 Gy), lethal (2 x 4 Gy), and supralethal (2 x 6 Gy) doses of radiation prior to transplantation. Lethal and supralethal doses of radiation differ in that the latter is fully myeloablative, whereas the former is only partially myeloablative. Sublethal radiation represents a model for nonmyeloablative conditioning regimens. Peripheral blood samples were obtained weekly for analysis of cellularity and for donor contributions to the three major blood lineages (Fig. 4, upper panels). The composition of the leukocyte compartment with respect to T lymphocytes, B lymphocytes, and myeloid cells was also determined for recipient, GFP donor, and CD45a donor populations (Fig. 4, lower panels).

Figure 4. A bone marrow transplant experiment in which equal numbers of bone marrow cells obtained from B6-GFP and B6-CD45a donors were transplanted into B6-hemoglobin ß (Hbb)d recipients after radiation conditioning at three doses. Top panels: Absolute cell numbers were derived by calculations based on complete blood counts and chimerism analysis for GFP, CD45a, and Hbbs. The horizontal lines in each plot indicate the lowest count found for that parameter in a group of 12 normal B6 mice. Normal distributions of each parameter (mean ¡À SD) in this group of mice were as follows: leukocytes, 8.8 ¡À 2.0 x 103 cells per µl; erythrocytes, 8.5 ¡À 0.6 x 106 cells per µl; platelets, 930 ¡À 270 x 103 cells per µl. Bottom panels: Peripheral blood leukocytes (WBC) were phenotyped by flow cytometry and are plotted as the number of cells in myeloid (CD11b/Ly-6G), T lymphocyte (CD4/CD8), and B lymphocyte (CD19) lineages that are derived from endogenous recovery or from each of the two donors of bone marrow cells, as indicated. Abbreviations: GFP, green fluorescent protein; WBC, white blood cells.
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Sublethal radiation clearly depleted the peripheral blood of leukocytes by 1 week and of platelets by 2 weeks. However, the eventual recovery of these lineages seen between 2 and 5 weeks post-transplantation was predominantly derived from endogenous stem cells rather than by transplanted bone marrow (Fig. 4, leukocytes and platelets). Erythrocyte counts were not affected by sublethal radiation, but Hbb analysis showed a clear contribution of transplanted bone marrow cells to the erythroid compartment at 2 and 4 weeks (Fig. 4, erythrocytes). Interestingly, G6-GFP donor-derived myeloid cells appeared transiently in very small numbers 2 weeks following sublethal radiation but were not detected at later times, and B6-GFP-derived lymphocytes were not detected at any time after transplantation. In contrast, the leukocytes derived from the B6-CD45a donor, although also present in small numbers, persisted from 2 to 5 weeks and included mostly B lymphocytes (Fig. 4, lower panels). After higher doses of radiation, contributions of the two donor populations to the leukocyte subset were comparable, with a slight predominance of B6-GFP-derived cells seen at 5 weeks post-transplant. Erythrocytes derived from the two bone marrow donors could not be distinguished from each other due to the lack of GFP expression in the erythroid lineage, but Hbb analysis clearly showed a dominance of donor-derived erythrocytes at 2 weeks that increased in number over subsequent weeks (Fig. 4, erythrocytes). Platelets derived from the B6-GFP donor were readily detectable beginning at 2 weeks, and by 5 weeks, over half of the platelets in peripheral blood of the high-dose radiation groups were GFPpos. Although platelets derived from the B6-CD45a donor could not be distinguished from those derived due to endogenous recovery, we presume that most of the GFPneg platelets seen 5 weeks after myeloablative radiation are derived from the B6-CD45a donor.; r: {; w  a9 W9 Z1 M. H
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The relative inefficiency of B6-GFP donor cells to engraft in 4-Gy-irradiated recipient mice was confirmed in a second experiment, in which B6-GFP and B6-CD45a donor cells were independently transplanted into separate groups of sublethally conditioned recipient mice. We observed transient, low-level contributions of both grafts to the erythroid lineage and sustained low-level engraftment of leukocytes in four of four recipients of CD45a donor cells. In contrast, three of four recipients of B6-GFP donor bone marrow exhibited little if any leukocyte engraftment, whereas one of four recipients engrafted in the leukocyte lineage (Fig. 5). These data demonstrate a lineage-selective effect of relative resistance to leukocyte engraftment by B6-GFP-derived donor cells under conditions of sublethal radiation and limited donor cell transplantation, suggesting that the resistance occurs after megakaryocyte and erythroid lineage divergence from the nucleated cell lineages.: M8 D/ s1 T  z( p; W

Figure 5. A bone marrow transplant experiment in which bone marrow cells obtained from B6-GFP and B6-CD45a donors were transplanted separately into B6-Hbbd recipients after radiation conditioning at a dose of 4 Gy. Top panel: Leukocyte (T cells, B cells, and myeloid cells) engraftment was detected by flow cytometric analysis after antibody staining of CD19, CD4, CD8, Mac-1, and Gr-1 antigens. The cell number was derived from donor contribution fractions from flow cytometric analysis and total leukocyte count. CD45a donor (circles) engraftment of leukocytes was significantly higher than GFP donor (filled squares) engraftment, excluding the GFP donor outlier (open squares). Bottom panel: Hemoglobin contributions from B6-GFP donor (Hdds) and B6-CD45a Donor (Hdds) bone marrow transplants into B6-Hbbd recipients after sublethal radiation conditioning (4 Gy). Donor-derived hemoglobin concentrations were calculated from total hemoglobin concentrations (g/dl), and the percentages of donor contribution as determined by HPLC analysis. The difference between the engraftment of CD45a donor (circles) and the engraftment of GFP donor (filled squares) was statistically insignificant, although one outlier from the GFP donor group (open squares) was observed to show a more successful engraftment. Abbreviation: Hbb, hemoglobin ß.+ {0 y3 [4 l$ u. n3 Z4 i7 l8 f
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We also evaluated the effect of sex matching in the sublethal radiation setting by transplanting a 50:50 mix of GFP-transgenic and CD45 congenic cells obtained from male donors into two groups of 4-Gy-irradiated recipients, male or female. We observed better engraftment of both B6-CD45a and B6-GFP cells when donor and host were sex-matched, but even in this case, the engraftment of B6-CD45a cells was on average 10-fold higher than B6-GFP cells. Furthermore, B6-CD45a cells engraftment was fivefold higher in the sex-mismatched combination compared with B6-GFP cells in the sex-matched combination (0.44 ¡À 0.15 x 103 cells per µl vs. 0.09 ¡À 0.01 x 103 cells per µl; p

DISCUSSION* K4 B8 ^( {9 `2 Q
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Many of the early studies documenting engraftment kinetics following bone marrow transplantation used Hbb as an indicator of donor-derived hematopoiesis . This study sheds new light on this old controversy by directly comparing erythroid, platelet, and leukocyte engraftment kinetics in an animal model of transplantation. Our results emphasize the fact that the erythroid lineage displays characteristics of engraftment that are distinct from those of leukocyte and platelet lineages, as shown by transient engraftment of erythrocytes in a sublethally irradiated setting, where little if any engraftment of leukocytes and platelets is seen (Fig. 4, upper panels).

Hbb variants are typically distinguished by gel electrophoresis, but clinical protocols that use HPLC have been introduced in recent years. We developed a protocol to allow distinction of mouse Hbb variants by HPLC as a rapid, sensitive, and quantitative method to measure chimerism in the erythroid lineage. This method uses an acidic derivatization of cysteine residues to introduce a differential charge between Hbbs and Hbbd. HPLC allows rapid, reproducible analyses of Hbb isoforms with precise quantitation of isoforms in an automated fashion, which facilitates the determination of graft-derived erythrocytes. Recently, a new transgenic mouse strain has been reported that retains GFP fluorescence in erythrocytes, providing a transplant model in which flow cytometry can be used to determine donor contributions to all blood lineages as well as in many other tissues  in nonmyeloablative transplant models (Figs. 4, 5). Therefore, our data support continued application of congenic systems rather than transgenic models in studies that aim to model engraftment in reduced intensity chemotherapy regimens. For routine analysis of Hbb alleles, the HPLC method reported here provides an attractive alternative to traditional gel electrophoresis.0 E8 V/ w( k( O3 A( `

DISCLOSURES* \! d5 ?% M1 p0 {1 u( I# [
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The authors indicate no potential conflicts of interest.
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ACKNOWLEDGMENTS4 B2 U. O1 m* B% c; _1 l# S
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This research was supported by National Institutes of Health Grants HL072026 (G.J.S.), DK57899 (G.J.S.), HL069133 (W.H.F.), HL077818 (W.H.F.), and T32 DK007115 (S.C.).  l& ]. g6 p2 f0 j+ K
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