Quantification of green fluorescent protein by in vivo imaging, PCR, and flow cytometry: Comparison of transgenic strains and relevance for fetal cell microchimerism

Abstract

Animal models are increasingly being used for the assessment of fetal cell microchimerism in maternal tissue. We wished to determine the optimal transgenic mouse strain and analytic technique to facilitate the detection of rare transgenic microchimeric fetal cells amongst a large number of maternal wild-type cells. We evaluated two strains of mice transgenic for the enhanced green fluorescent protein (EGFP): a commercially available, commonly used strain (C57BL/6-Tg(ACTB-EGFP)10sb/J) (CAG) and a newly created strain (ROSA26-EGFP) using three different techniques: in vivo and ex vivo fluorescent imaging (for whole body and dissected organs, respectively), PCR amplification of gfp, and flow cytometry (FCM). By fluorescent imaging, organs from CAG mice were 10-fold brighter than organs from ROSA26-EGFP mice (P < 0.0001). By PCR, more transgene from CAG mice was detected compared to ROSA26-EGFP mice (P 5 0.04). By FCM, ROSA26-EGFP cell fluorescence was more uniform than CAG cells. A greater proportion of cells from ROSA26-EGFP organs were positive for EGFP than cells from CAG organs, but CAG mice had a greater proportion of cells with the brightest fluorescent intensity. Each transgenic strain possesses characteristics that make it useful under specific experimental circumstances. The CAG mouse model is preferable when experiments require brighter cells, whereas ROSA26-EGFP is more appropriate when uniform or ubiquitous expression is more important than brightness. Investigators must carefully select the transgenic strain most suited to the experimental design to obtain the most consistent and reproducible data. In vivo imaging allows for phenotypic evaluation of whole animals and intact organs; however, we did not evaluate its utility for the detection of rare, fetal microchimeric cells in the maternal organs. Finally, while PCR amplification of a paternally inherited transgene does allow for the quantitative determination of rare microchimeric cells, FCM allows for both quantitative and qualitative evaluations of fetal cells at very high sensitivity in a plethora of maternal organs. '

Fetomaternal Trafficking in the Mouse Increases as Delivery Approaches and Is Highest in the Maternal Lung1

“…More recently, we compared methods (e.g., flow cytometry [FCM] and real-time PCR) to detect rare transgenic fetal cells using a paternally inherited enhanced green fluorescence protein (Gfp) transgene sequence as a fetal marker in the wild-type C57BL/6J pregnant female [18]. The Gfp sequence is transmitted as a hemizygous dominantly expressed gene.…”

“…This allows for the localization of fetal cells in maternal tissues during pregnancy and postpartum. Flow cytometry has the capability to analyze whole organs or sort live cells for further evaluation with the highest sensitivity and specificity [18]. The technique is less time consuming than histochemical evaluation, therefore allowing for the analysis of multiple tissue types at multiple time points.…”

Multiparity improves outcomes after cerebral ischemia in female mice despite features of increased metabovascular risk

“…The majority of fetal microchimerism studies have examined the presence and to a lesser extent, the function, of MCs. Although studies indicate the maternal brain has the lowest frequency of fetal microchimerism of all organs (39,40), MCs have been found in the CNS in both healthy and diseased humans and mice (41). Indeed, the rarity of these cells precluded our efforts to perform quantification and more comprehensive phenotypic analysis.…”

Comprehensive Analysis of Genes Expressed by Rare Microchimeric Fetal Cells in the Maternal Mouse Lung1

“…In mice and humans, fetal cells enter the maternal circulation early in pregnancy, increase throughout gestation, and sharply decrease in the days following delivery [1,2].…”