Duplex Polymerase Chain Reaction Quantification of Human Cells in a Murine Background
http://www.100md.com
《干细胞学杂志》
a Laboratory of Molecular Medicine, Interdisciplinary Centre for Clinical Research, University of Leipzig Faculty of Medicine, Germany;
b In Vitro Differentiation Group, IPK, Gatersleben, Germany;
c Institute for Immunology, University of Leipzig Faculty of Medicine, Germany;
d Vita 34 AG, Leipzig, Germany
Key Words. Species-specific polymerase chain reaction ? Human ? Mouse ? Chimera ? Stem cell ? Metastasis ? Quantification
Correspondence: Michael Cross, Ph.D., Division of Hematology/Oncology, University of Leipzig, Inselstrasse 22, 04103 Leipzig, Germany. Telephone: 49-0-341-97-15942; Fax: 49-0-341-97-15979; e-mail: crossm@medizin.uni-leipzig.de
ABSTRACT
The transplantation of human cells into immune-deficient mice is an increasingly common approach in studies of tumor progression and studies of the in vivo regenerative potential of human stem cells . The quality of the information derived from these models depends on the accuracy with which low numbers of donor-derived cells can be detected in host tissues, and a variety of methods have been developed to this end. These include the use of species-specific antibodies or genetic tags such as ?-galactosidase , luciferase , or fluorescent protein genes to highlight cells in tissue sections, in cell suspensions, and even in living animals . Although cell imaging techniques can provide detailed information down to the single-cell level in a defined location , they are not well suited to the widespread and systematic quantitation of low-level contribution throughout a range of tissues and organs.
Species-specific polymerase chain reaction (PCR) offers a more effective means of detecting and quantifying low levels of donor contribution under these conditions, particularly when using primers to repetitive rather than single-copy target sequences. Hence, a primer pair specific to the -satellite repeat of human chromosome 17 has been used to detect low levels of human hematopoiesis in xenotransplanted nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mice , whereas a real-time PCR modification of this procedure allows accurate quantitation down to 1 in 105 to 106 cells . However, the exclusive use of human primers in these cases can limit the practical applications. First, accurate quantitation requires absolute consistency in the purity and amount of DNA introduced into the reaction. This can be difficult to achieve, particularly when comparing samples extracted from a range of different organs under different conditions. Second, the methods require the use of real-time PCR analysis, limiting their use to laboratories with direct access to this technology.
With the aim of providing a simple, sensitive, and reliable means of human cell quantification in murine tissues, we have developed a duplex PCR approach involving the coamplification of murine and human repetitive sequences and found that a single reaction condition provides quantification of the ratio of human-to-mouse DNA over a wide range without the need for real-time PCR. The method has been used to analyze xenotrans-planted mice and an in vitro model of human stem cell integration into hanging drop cultures of murine embryonal stem (ES) cells, demonstrating superior contribution by the stem cell–enriched CD133+ fraction.
MATERIALS AND METHODS
Primer Design and Species Specificity
Our aim was to establish a duplex PCR assay that provides a measure of the ratio of human-to-mouse DNA over a range of experimental conditions in which the human component is widely variable but generally low and the amount of material available for assay is often limiting. For the sensitive and specific detection of human cells, we used previously described primers that amplify a major product of approximately 1,000 bp (with minor products of approximately 680 and 340 bp) from the highly repetitive alpha satellite repeats of human chromosome 7 . These tandem repeats are present at many thousands of copies per cell and are highly species-specific. To complement the human alpha satellite target, we designed primers to a 728-bp section of a mouse-specific repetitive sequence that is present at 60 to 100 copies on chromosome 8 . The coamplification of a highly repetitive human and a middle-repetitive murine target sequence provides the high level of sensitivity required for the routine processing of small samples while biasing the relative sensitivity of a duplex PCR toward the detection of human cells. Furthermore, the murine primers were designed to have melting temperatures significantly lower than those of the human primers, to allow further adjustment of the relative reaction efficiencies towards one or other species by choosing an appropriate annealing temperature (see below).
To confirm the effects of annealing temperature, a standard preparation of 0.5% human-in-mouse DNA was amplified with the human and mouse primer sets both alone and in combination in a temperature-gradient PCR using annealing temperatures between 53°C and 63°C (Fig. 1). When used alone, the human primers generated the expected products consistently over the entire range of annealing temperatures, with no evidence of nonspecific products at the less-stringent lower temperatures. The murine primers performed well up to an annealing temperature of 62°C, above which the product yield decreased. In the presence of both primer pairs, higher annealing temperatures favored the human reaction and further reduced the yield of mouse product, whereas the lower annealing temperatures favored the murine reaction and reduced the final yield of human product. This apparent competition effect most likely occurs as a consequence of the feedback inhibition or substrate depletion, which marks the end of the logarithmic phase of PCR amplification. In the case of a duplex PCR, this is triggered by the total sum of products, regardless of which of the two reactions (human or mouse) has been most productive.
Figure 1. The effects of annealing temperature on amplification efficiency. Amplification products from temperature-gradient poly-merase chain reactions of 0.5% human-in-mouse DNA using primers specific for (A) human chromosome 7 -satellite sequences, (B) mouse chromosome 8 centromeric repeat sequence, and (C) a mixture of both primer pairs.
Sensitivity and Range of Quantification
The competition effect apparent from Figure 1 suggests that a duplex PCR reaction may on the one hand be less sensitive than a human-only reaction to very low levels of human DNA but on the other hand may provide quantitation (in terms of the ratio of human-to-murine products) over a much wider range. The limits of detection and range of quantitation of the two approaches were therefore compared directly using samples taken from the same dilution series (Fig. 2). The human-only reaction produced a weak signal from 0.001% human-in-mouse DNA after 35 cycles, and the product yield was increased further by extending the reaction to 40 cycles. In comparison, the duplex reaction detected human-in-mouse down to 0.01% at 35 cycles. Since the duplex reaction accumulates more overall product and reaches an end point by this stage, the detection limit is not extended by further cycles (not shown). Under our standard conditions, the detection limit of the duplex reaction therefore lies between 0.001% and 0.01% human-in-mouse and is up to 10-fold less sensitive than the human-only reaction.
Figure 2. The limits of detection and range of quantification under various reaction conditions. Samples prepared from titrated mixtures of human and mouse cells were amplified using (A) human primers only at an annealing temperature of 59°C or (B) a duplex mixture of human and mouse primers under conditions that bias toward the murine (annealing at 53°C) or human (annealing at 59°C) products. After electrophoresis and image analysis, the product yield of the (C) human-only polymerase chain reaction (PCR) and the (D) human-to-mouse product ratio of the duplex PCR were plotted against the input ratio on double logarithmic scales to show the relationship over the entire range.
However, the plots of product yield (human-only PCR) or product ratio (duplex PCR) against input DNA reveal a major advantage of the duplex approach, in that the product ratios reflect closely those of the input target DNA over the entire range, from the lowest limit of detection up to 50% human content. Once again, this most likely occurs as a result of either feedback inhibition or substrate exhaustion, which conserves the product ratios at the end of the logarithmic phase of amplification. Regardless of the cycle number at which this point is reached, the information is therefore maintained throughout the subsequent plateau phase. The end-stage analysis of a single PCR reaction condition therefore suffices to yield a measure of human-in-mouse content from 0.01% up to >50%.
Analysis of Transplanted Mice and Human/Mouse Cocultures
Having established the sensitivity and range of quantitation using titrated DNAs, the duplex PCR method was used to analyze two relevant experimental models. First, spleen tissue from four independent NOD/SCID mice transplanted with human cord blood–derived mononuclear cells was analyzed both by FACS and by duplex PCR. To enable statistical analysis, the PCR reactions were performed in triplicate under double-blind conditions. As shown in Figure 3A, the results of the duplex PCR were highly reproducible and in close agreement with those of FACS analysis.
Figure 3. Duplex PCR analysis of chimeric mice and cultures. (A): PCR reactions were performed in triplicate (PCR #1-3) using spleen tissue from four transplanted mice (1–4). Mean and standard deviations are shown for the two spleens that fell within the assay range of 0.01%–50% human contribution. The corresponding coefficients of variation were 16.1% (mouse 3) and 11.8% (mouse 4). (B): Mixed cultures of CD133+ or CD133– human umbilical cord blood cells (5%) and mouse ES cells (95%) were established using either undifferentiated ES cells or ES-derived cells from various stages of a pancreatic differentiation culture (5 plus 16, 5 plus 27, and 5 plus 30 days). After an additional 5 days of coculture, cell lysates were analyzed by duplex PCR (35 cycles, annealing at 59°C). The percent human DNA content of mixed cultures calculated from the scanned gels is shown above each lane. Standard: DNA standards run in parallel. Abbreviations: ES, embryonal stem; FACS, fluorescence-activated cell sorter; PCR, polymerase chain reaction.
One of the original reasons for developing a duplex PCR based on repetitive sequences was to facilitate the rapid and reliable screening of large numbers of small-scale cocultures of murine and human cells. In one such approach, cells purified from human umbilical cord blood were mixed at a frequency of 5% either with undifferentiated mouse ES cells or with ES-derived cells from various stages of a pancreatic differentiation . After 5 days of coculture, individual cultures were harvested and cell lysates were introduced directly into the duplex PCR assay. The results (Fig. 3B) show that whereas CD133+ human cord blood cells are capable of making a detectable contribution to hanging drop cultures of both undifferentiated and differentiating ES cells, the CD133– population contributes relatively poorly to EBs established from undifferentiated ES cells and only rarely to those established from the later stages of pancreatic differentiation.
DISCUSSION
We describe a duplex PCR technique in which human and murine-specific, repetitive sequences are coamplified in a single reaction. Determination of the ratio of human-to-mouse products rather than the absolute amount of human material compensates for variations arising during sample preparation. Because the proportion of human-to-mouse products is maintained into the plateau phase of the PCR reaction, there is no need to use real-time PCR technology, a single reaction being sufficient to quantify human contribution from <0.01% to >50%. This provides a simple, sensitive, and reliable indication of the frequency of human cells in a murine background that should be of widespread use to studies of human stem cell potential both in vivo and in vitro. In this study, the technique has been used to quantify human cells in xenotransplanted mice and to demonstrate the contribution of human CD133+ cord blood stem cells to differentiation cultures established from murine ES cells.
ACKNOWLEDGMENTS
Visonneau S, Cesano A, Torosian MH et al. Growth characteristics and metastatic properties of human breast cancer xenografts in immunodeficient mice. Am J Pathol 1998;152:1299–1311.
Dick JE, Bhatia M, Gan O et al. Assay of human stem cells by repopulation of NOD/SCID mice. STEM CELLS 1997;15:199–203.
Toma C, Pittenger MF, Cahill KS et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002;105:93–98.
Wang X, Rosol M, Ge S et al. Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging. Blood 2003;102:3478–3482.
Tamaki S, Eckert K, He D et al. Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. J Neurosci Res 2002;69:976–986.
Jenkins DE, Oei Y, Hornig YS et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin Exp Metastasis 2003;20:733–744.
Yamamoto N, Yang M, Jiang P et al. Real-time GFP imaging of spontaneous HT-1080 fibrosarcoma lung metastases. Clin Exp Metastasis 2003;20:181–185.
Panoskaltsis-Mortari A, Price A, Hermanson JR et al. In vivo imaging of graft-versus-host-disease in mice. Blood 2004;103:3590–3598.
Yang M, Baranov E, Wang JW et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model. Proc Natl Acad Sci U S A 2002;99:3824–3829.
Warburton PE, Greig GM, Haaf T et al. PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 1991;11:324–333.
Mobest D, Goan SR, Junghahn I etal. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. STEM CELLS 1999;17:152–161.
Becker M, Nitsche A, Neumann C et al. Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems. Br J Cancer 2002:87:1328–1335.
Spooncer E, Heyworth CM, Dunn A et al. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation 1986;31:111–118.
Koeffler HP, Golde DW. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science 1978;200:1153–1154.
Nagy A, Rossant J, Nagy R et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 1993;90:8424–8428.
Wobus AM, Guan K, Yang H-T et al. Embryonic stem cells as a model to study cardiac, skeletal muscle and vascular smooth muscle cell differentiation. Methods Mol Biol 2002;185:127–156.
Kania G, Blyszczuk P, Czyz J et al. Differentiation of mouse embryonic stem cells into pancreatic and hepatic cells. Methods Enzymol 2003;365:287–303.
Blyszczuk P, Czyz J, Kania G et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci U S A 2003;100:998–1003.
Boyle AL, Ward DC. Isolation and initial characterization of a large repeat sequence element specific to mouse chromosome 8. Genomics 1992;12:517–525.
Nitsche A, Becker M, Junghahn I et al. Quantification of human cells in NOD/SCID mice by duplex real-time polymerase-chain reaction. Haematologica 2001;86:693–699.(Oliver Pelza, Minyao Wua,)
b In Vitro Differentiation Group, IPK, Gatersleben, Germany;
c Institute for Immunology, University of Leipzig Faculty of Medicine, Germany;
d Vita 34 AG, Leipzig, Germany
Key Words. Species-specific polymerase chain reaction ? Human ? Mouse ? Chimera ? Stem cell ? Metastasis ? Quantification
Correspondence: Michael Cross, Ph.D., Division of Hematology/Oncology, University of Leipzig, Inselstrasse 22, 04103 Leipzig, Germany. Telephone: 49-0-341-97-15942; Fax: 49-0-341-97-15979; e-mail: crossm@medizin.uni-leipzig.de
ABSTRACT
The transplantation of human cells into immune-deficient mice is an increasingly common approach in studies of tumor progression and studies of the in vivo regenerative potential of human stem cells . The quality of the information derived from these models depends on the accuracy with which low numbers of donor-derived cells can be detected in host tissues, and a variety of methods have been developed to this end. These include the use of species-specific antibodies or genetic tags such as ?-galactosidase , luciferase , or fluorescent protein genes to highlight cells in tissue sections, in cell suspensions, and even in living animals . Although cell imaging techniques can provide detailed information down to the single-cell level in a defined location , they are not well suited to the widespread and systematic quantitation of low-level contribution throughout a range of tissues and organs.
Species-specific polymerase chain reaction (PCR) offers a more effective means of detecting and quantifying low levels of donor contribution under these conditions, particularly when using primers to repetitive rather than single-copy target sequences. Hence, a primer pair specific to the -satellite repeat of human chromosome 17 has been used to detect low levels of human hematopoiesis in xenotransplanted nonobese diabetic/ severe combined immunodeficiency (NOD/SCID) mice , whereas a real-time PCR modification of this procedure allows accurate quantitation down to 1 in 105 to 106 cells . However, the exclusive use of human primers in these cases can limit the practical applications. First, accurate quantitation requires absolute consistency in the purity and amount of DNA introduced into the reaction. This can be difficult to achieve, particularly when comparing samples extracted from a range of different organs under different conditions. Second, the methods require the use of real-time PCR analysis, limiting their use to laboratories with direct access to this technology.
With the aim of providing a simple, sensitive, and reliable means of human cell quantification in murine tissues, we have developed a duplex PCR approach involving the coamplification of murine and human repetitive sequences and found that a single reaction condition provides quantification of the ratio of human-to-mouse DNA over a wide range without the need for real-time PCR. The method has been used to analyze xenotrans-planted mice and an in vitro model of human stem cell integration into hanging drop cultures of murine embryonal stem (ES) cells, demonstrating superior contribution by the stem cell–enriched CD133+ fraction.
MATERIALS AND METHODS
Primer Design and Species Specificity
Our aim was to establish a duplex PCR assay that provides a measure of the ratio of human-to-mouse DNA over a range of experimental conditions in which the human component is widely variable but generally low and the amount of material available for assay is often limiting. For the sensitive and specific detection of human cells, we used previously described primers that amplify a major product of approximately 1,000 bp (with minor products of approximately 680 and 340 bp) from the highly repetitive alpha satellite repeats of human chromosome 7 . These tandem repeats are present at many thousands of copies per cell and are highly species-specific. To complement the human alpha satellite target, we designed primers to a 728-bp section of a mouse-specific repetitive sequence that is present at 60 to 100 copies on chromosome 8 . The coamplification of a highly repetitive human and a middle-repetitive murine target sequence provides the high level of sensitivity required for the routine processing of small samples while biasing the relative sensitivity of a duplex PCR toward the detection of human cells. Furthermore, the murine primers were designed to have melting temperatures significantly lower than those of the human primers, to allow further adjustment of the relative reaction efficiencies towards one or other species by choosing an appropriate annealing temperature (see below).
To confirm the effects of annealing temperature, a standard preparation of 0.5% human-in-mouse DNA was amplified with the human and mouse primer sets both alone and in combination in a temperature-gradient PCR using annealing temperatures between 53°C and 63°C (Fig. 1). When used alone, the human primers generated the expected products consistently over the entire range of annealing temperatures, with no evidence of nonspecific products at the less-stringent lower temperatures. The murine primers performed well up to an annealing temperature of 62°C, above which the product yield decreased. In the presence of both primer pairs, higher annealing temperatures favored the human reaction and further reduced the yield of mouse product, whereas the lower annealing temperatures favored the murine reaction and reduced the final yield of human product. This apparent competition effect most likely occurs as a consequence of the feedback inhibition or substrate depletion, which marks the end of the logarithmic phase of PCR amplification. In the case of a duplex PCR, this is triggered by the total sum of products, regardless of which of the two reactions (human or mouse) has been most productive.
Figure 1. The effects of annealing temperature on amplification efficiency. Amplification products from temperature-gradient poly-merase chain reactions of 0.5% human-in-mouse DNA using primers specific for (A) human chromosome 7 -satellite sequences, (B) mouse chromosome 8 centromeric repeat sequence, and (C) a mixture of both primer pairs.
Sensitivity and Range of Quantification
The competition effect apparent from Figure 1 suggests that a duplex PCR reaction may on the one hand be less sensitive than a human-only reaction to very low levels of human DNA but on the other hand may provide quantitation (in terms of the ratio of human-to-murine products) over a much wider range. The limits of detection and range of quantitation of the two approaches were therefore compared directly using samples taken from the same dilution series (Fig. 2). The human-only reaction produced a weak signal from 0.001% human-in-mouse DNA after 35 cycles, and the product yield was increased further by extending the reaction to 40 cycles. In comparison, the duplex reaction detected human-in-mouse down to 0.01% at 35 cycles. Since the duplex reaction accumulates more overall product and reaches an end point by this stage, the detection limit is not extended by further cycles (not shown). Under our standard conditions, the detection limit of the duplex reaction therefore lies between 0.001% and 0.01% human-in-mouse and is up to 10-fold less sensitive than the human-only reaction.
Figure 2. The limits of detection and range of quantification under various reaction conditions. Samples prepared from titrated mixtures of human and mouse cells were amplified using (A) human primers only at an annealing temperature of 59°C or (B) a duplex mixture of human and mouse primers under conditions that bias toward the murine (annealing at 53°C) or human (annealing at 59°C) products. After electrophoresis and image analysis, the product yield of the (C) human-only polymerase chain reaction (PCR) and the (D) human-to-mouse product ratio of the duplex PCR were plotted against the input ratio on double logarithmic scales to show the relationship over the entire range.
However, the plots of product yield (human-only PCR) or product ratio (duplex PCR) against input DNA reveal a major advantage of the duplex approach, in that the product ratios reflect closely those of the input target DNA over the entire range, from the lowest limit of detection up to 50% human content. Once again, this most likely occurs as a result of either feedback inhibition or substrate exhaustion, which conserves the product ratios at the end of the logarithmic phase of amplification. Regardless of the cycle number at which this point is reached, the information is therefore maintained throughout the subsequent plateau phase. The end-stage analysis of a single PCR reaction condition therefore suffices to yield a measure of human-in-mouse content from 0.01% up to >50%.
Analysis of Transplanted Mice and Human/Mouse Cocultures
Having established the sensitivity and range of quantitation using titrated DNAs, the duplex PCR method was used to analyze two relevant experimental models. First, spleen tissue from four independent NOD/SCID mice transplanted with human cord blood–derived mononuclear cells was analyzed both by FACS and by duplex PCR. To enable statistical analysis, the PCR reactions were performed in triplicate under double-blind conditions. As shown in Figure 3A, the results of the duplex PCR were highly reproducible and in close agreement with those of FACS analysis.
Figure 3. Duplex PCR analysis of chimeric mice and cultures. (A): PCR reactions were performed in triplicate (PCR #1-3) using spleen tissue from four transplanted mice (1–4). Mean and standard deviations are shown for the two spleens that fell within the assay range of 0.01%–50% human contribution. The corresponding coefficients of variation were 16.1% (mouse 3) and 11.8% (mouse 4). (B): Mixed cultures of CD133+ or CD133– human umbilical cord blood cells (5%) and mouse ES cells (95%) were established using either undifferentiated ES cells or ES-derived cells from various stages of a pancreatic differentiation culture (5 plus 16, 5 plus 27, and 5 plus 30 days). After an additional 5 days of coculture, cell lysates were analyzed by duplex PCR (35 cycles, annealing at 59°C). The percent human DNA content of mixed cultures calculated from the scanned gels is shown above each lane. Standard: DNA standards run in parallel. Abbreviations: ES, embryonal stem; FACS, fluorescence-activated cell sorter; PCR, polymerase chain reaction.
One of the original reasons for developing a duplex PCR based on repetitive sequences was to facilitate the rapid and reliable screening of large numbers of small-scale cocultures of murine and human cells. In one such approach, cells purified from human umbilical cord blood were mixed at a frequency of 5% either with undifferentiated mouse ES cells or with ES-derived cells from various stages of a pancreatic differentiation . After 5 days of coculture, individual cultures were harvested and cell lysates were introduced directly into the duplex PCR assay. The results (Fig. 3B) show that whereas CD133+ human cord blood cells are capable of making a detectable contribution to hanging drop cultures of both undifferentiated and differentiating ES cells, the CD133– population contributes relatively poorly to EBs established from undifferentiated ES cells and only rarely to those established from the later stages of pancreatic differentiation.
DISCUSSION
We describe a duplex PCR technique in which human and murine-specific, repetitive sequences are coamplified in a single reaction. Determination of the ratio of human-to-mouse products rather than the absolute amount of human material compensates for variations arising during sample preparation. Because the proportion of human-to-mouse products is maintained into the plateau phase of the PCR reaction, there is no need to use real-time PCR technology, a single reaction being sufficient to quantify human contribution from <0.01% to >50%. This provides a simple, sensitive, and reliable indication of the frequency of human cells in a murine background that should be of widespread use to studies of human stem cell potential both in vivo and in vitro. In this study, the technique has been used to quantify human cells in xenotransplanted mice and to demonstrate the contribution of human CD133+ cord blood stem cells to differentiation cultures established from murine ES cells.
ACKNOWLEDGMENTS
Visonneau S, Cesano A, Torosian MH et al. Growth characteristics and metastatic properties of human breast cancer xenografts in immunodeficient mice. Am J Pathol 1998;152:1299–1311.
Dick JE, Bhatia M, Gan O et al. Assay of human stem cells by repopulation of NOD/SCID mice. STEM CELLS 1997;15:199–203.
Toma C, Pittenger MF, Cahill KS et al. Human mesenchymal stem cells differentiate to a cardiomyocyte phenotype in the adult murine heart. Circulation 2002;105:93–98.
Wang X, Rosol M, Ge S et al. Dynamic tracking of human hematopoietic stem cell engraftment using in vivo bioluminescence imaging. Blood 2003;102:3478–3482.
Tamaki S, Eckert K, He D et al. Engraftment of sorted/expanded human central nervous system stem cells from fetal brain. J Neurosci Res 2002;69:976–986.
Jenkins DE, Oei Y, Hornig YS et al. Bioluminescent imaging (BLI) to improve and refine traditional murine models of tumor growth and metastasis. Clin Exp Metastasis 2003;20:733–744.
Yamamoto N, Yang M, Jiang P et al. Real-time GFP imaging of spontaneous HT-1080 fibrosarcoma lung metastases. Clin Exp Metastasis 2003;20:181–185.
Panoskaltsis-Mortari A, Price A, Hermanson JR et al. In vivo imaging of graft-versus-host-disease in mice. Blood 2004;103:3590–3598.
Yang M, Baranov E, Wang JW et al. Direct external imaging of nascent cancer, tumor progression, angiogenesis, and metastasis on internal organs in the fluorescent orthotopic model. Proc Natl Acad Sci U S A 2002;99:3824–3829.
Warburton PE, Greig GM, Haaf T et al. PCR amplification of chromosome-specific alpha satellite DNA: definition of centromeric STS markers and polymorphic analysis. Genomics 1991;11:324–333.
Mobest D, Goan SR, Junghahn I etal. Differential kinetics of primitive hematopoietic cells assayed in vitro and in vivo during serum-free suspension culture of CD34+ blood progenitor cells. STEM CELLS 1999;17:152–161.
Becker M, Nitsche A, Neumann C et al. Sensitive PCR method for the detection and real-time quantification of human cells in xenotransplantation systems. Br J Cancer 2002:87:1328–1335.
Spooncer E, Heyworth CM, Dunn A et al. Self-renewal and differentiation of interleukin-3-dependent multipotent stem cells are modulated by stromal cells and serum factors. Differentiation 1986;31:111–118.
Koeffler HP, Golde DW. Acute myelogenous leukemia: a human cell line responsive to colony-stimulating activity. Science 1978;200:1153–1154.
Nagy A, Rossant J, Nagy R et al. Derivation of completely cell culture-derived mice from early-passage embryonic stem cells. Proc Natl Acad Sci U S A 1993;90:8424–8428.
Wobus AM, Guan K, Yang H-T et al. Embryonic stem cells as a model to study cardiac, skeletal muscle and vascular smooth muscle cell differentiation. Methods Mol Biol 2002;185:127–156.
Kania G, Blyszczuk P, Czyz J et al. Differentiation of mouse embryonic stem cells into pancreatic and hepatic cells. Methods Enzymol 2003;365:287–303.
Blyszczuk P, Czyz J, Kania G et al. Expression of Pax4 in embryonic stem cells promotes differentiation of nestin-positive progenitor and insulin-producing cells. Proc Natl Acad Sci U S A 2003;100:998–1003.
Boyle AL, Ward DC. Isolation and initial characterization of a large repeat sequence element specific to mouse chromosome 8. Genomics 1992;12:517–525.
Nitsche A, Becker M, Junghahn I et al. Quantification of human cells in NOD/SCID mice by duplex real-time polymerase-chain reaction. Haematologica 2001;86:693–699.(Oliver Pelza, Minyao Wua,)