The purpose of these experiments was to determine the most useful transgenic animal model and analytical technique to apply to studies of fetal cell microchimerism. The low frequency of fetal cells that are retained in maternal tissues following pregnancy necessitates the most efficient and powerful system to analyze these cells, with a high signal to background noise ratio. Therefore, we compared two transgenic strains of EGFP+ mice using three different analytical techniques at whole body, organ and cellular levels. While the results demonstrated that neither transgenic strain is preferable to the other for all techniques studied, we show instead that each strain possesses characteristics that makes it useful under specific experimental circumstances, such as the analytical tool being used or the organs of interest to be analyzed. Therefore, an investigator must carefully select the transgenic strain that is most suited to the experimental design in order to obtain the most consistent and reproducible data. However, imaging of fluorescent cells in live animals is currently limited to labeled cells in blood vessels or in regions of known location (i.e. area of injection of cells)(12
). Therefore, due to the existing status of this imaging technology and the purely quantitative nature of PCR amplification, flow cytometry provides the best overall approach for the quantitative and qualitative evaluation of rare transgenic microchimeric fetal cells.
For in vivo imaging, whole body fluorescence is brighter in CAG (black) mice compared to ROSA26-EGFP (white) mice after shaving fur. Due to reflection and absorption of the excitation light used for imaging in animals with white and black coat colors, respectively, all animals regardless of color should be shaved to minimize false positive or negative results and to create uniform circumstances for the visualization of real fluorescent signals. Shaving will also increase the likelihood of visualizing transgenic, microchimeric fetal cell populations in a wild-type mother.
Using ex vivo
imaging, while each organ autofluoresces resulting in baseline photon count differences between organs, all organs are brighter in CAG mice compared to ROSA26-EGFP mice (with the exception of spleen). There are several possible reasons for the differences seen in fluorescent intensity of organs between the two strains. These include differences in transgene expression inherent to each strain or due to unique promoters, or due to different numbers of transgene copies incorporated into each strain. With respect to spleen, photon counts are higher in wild-type mice compared to transgenic mice, and spleen is the only organ in which fluorescence is brighter in ROSA26-EGFP compared to CAG mice. The reasons for these differences are unclear. However, since the spleen contains a large and perhaps variable number of cells from the erythroid lineage with reduced or absent transgene expression compared to other nucleated cells, fluorescent emission would be similarly affected. One solution may be the incorporation of spectral deconvolution into the methodology described here, leading to improvement of imaging quality through the highly specific discrimination between overlapping fluorescent signals (i.e. real and autofluorescence)(10
Because both CAG and ROSA26-EGFP mice have the same EGFP transgene sequence, the same pair of primers and fluorescently labeled probe was used for amplifying the target sequence by real-time PCR. The transgene was amplified in all organs analyzed. PCR amplification occurs at an earlier cycle threshold in some organs (such as kidney and heart) than other organs (such as thymus and blood). The quantity of the PCR product of CAG mice was 1.5 to 3 fold higher than ROSA26-EGFP with only one exception, blood. The reasons for the organ to organ differences are unclear. However, the copy number of the transgene may be higher in CAG mice than in ROSA26-EGFP. There may also be different insertion sites of the two transgenes. PCR has higher specificity than fluorescent imaging, as PCR of the EGFP sequence is always negative in all organs from wild type mice.
Overall, a greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs as determined by FCM, particularly in blood, bone marrow, spleen and thymus. This is agreement with the results of Giel-Moloney et al. (8
), who showed that greater than 90% of B220+ splenocytes, CD4+ and CD8+ thymocytes and CD11b+ bone marrow myeloid cells from ROSA26-EGFP bone marrow transplant recipients were EGFP positive over background strain levels. This was in contrast to levels in CAG recipients, where these levels were ~90%, 50% and 65%, respectively (8
). However, our results showed that the highest level of transgene expression in these organs, represented by maximum EGFP intensity above background fluorescence, occurs in cells from CAG mice. In addition, organs from CAG mice were brighter in peak fluorescence compared to those from ROSA26-EGFP in blood, spleen, liver, heart, kidney and brain. Interestingly, peak fluorescence of bone marrow, thymus and lung tissue is higher in ROSA26-EGFP mice than CAG. The reasons for these organ differences are unclear. In addition, there appear to be differences in fluorescence intensity from animal to animal, as demonstrated by differences in EGFP intensity of cells from bone marrow. These differences could be due to technical variation, or could be due to biological factors, such as differences in transgene expression that are influenced by parental imprinting (13
). Nevertheless, blood cells from ROSA26-EGFP mice exhibit fairly uniform EGFP expression, which may make this model useful for studies of hematopoietic chimerism. Conversely, cells from CAG organs exhibit a wider range of transgene expression than ROSA26-EGFP mice, from very bright to very dim. There was also a considerable number of cells that showed no EGFP expression in CAG mice. Overall, the expression level of EGFP in ROSA26-EGFP is much more consistent between each organ than CAG mice, even though the peak fluorescence levels were lower in ROSA26-EGFP mice.
As with blood, the level of fluorescence of liver, heart, lung and kidney from ROSA26-EGFP mice is highly consistent, but the fluorescence is lower than that in CAG mice. Among cells with variable transgene expression in ROSA26-EGFP (e.g. those from spleen and bone marrow), it is not possible to distinguish between transgenic cells with a low level of transgene expression and wild-type cells. Overall, the CAG mouse model is useful when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Further characterization of both EGFP positive and negative cell types following breeding of transgenic males and wild type females will be necessary to continue to contribute to the understanding of the dynamics of maternal-fetal cell trafficking. This includes an assessment of the phenotype of fluorescently negative cells from specific transgenic organs, such as lung, to determine why a relatively high subset does not fluoresce. It is possible that the difference in fluorescent intensity among transgenic cell types in various organs is due to EGFP promoter specificity. Nevertheless, the aim of the current study was to subjectively assess multiple strains and methodologies for the detection of fetal transgenic DNA sequences or their corresponding RNA transcripts and protein products to more fully understand the biology of fetal maternal cell trafficking. Additional experiments are undoubtedly necessary using combined analytical approaches (e.g. flow cytometry and immunohistochemistry) to continue to understand this phenomenon. These experiments also include the assessment of bright, far-red fluorescent proteins, such as the far-red mutant TurboFP635 (14
), and DsRed mouse strains that are currently commercially available, such as B6.Cg-Tg(ACTB-Bgeo,-DsRed*MST)1Nagy/J. These new and highly innovative vectors may allow for improved sensitivity and specificity due to better tissue penetration, increased signal to noise ratios through the reduction of autofluorescence. However, for the current study we selected mouse strains that are transgenic for green fluorescent protein as these are the animals for which we have the most experience and which have been most commonly used as animal models of fetal cell microchimerism.
The aim of this study was to determine the potential usefulness of the newly created ROSA26-EGFP transgenic mouse strain compared to the widely used strain of mice transgenic for EGFP, CAG. The very low frequency of microchimeric cells in the host organs means that the selection of an optimal transgenic mouse strain and methods of transgene and/or transgenic cell detection are essential for the acquisition of consistent and reproducible data. We show here that in vivo
imaging, PCR and FCM possess differences in sensitivity and specificity between strains of transgenic mice. Our results suggest that sensitivity and specificity of these techniques are mutually exclusive when used for rare cell detection. For higher specificity, the brighter peak fluorescence intensity of cells from CAG mice give this model a definitive advantage to distinguish true signals from background. In contrast, ROSA26-EGFP mice have the advantage of sensitivity, as the majority of transgenic cells have ubiquitous EGFP expression. Regarding methodology, while cellular and subcellular resolution using in vivo
imaging techniques is possible, reports to date have only described its use in the detection of labeled cells in blood vessels or areas of known location (e.g. region of injection of cells)(12
), not for cells of unknown location deep in solid tissue. While further development of this imaging technology may allow for the localization of rare fetal microchimeric cells, currently the ability of flow cytometry to provide for highly sensitive quantitative and qualitative evaluations of rare transgenic cells suggests it is the most versatile and useful method for studies of fetal cell microchimerism and should be considered as the primary tool for these investigations.