Quantitative Analysis of Male Fetal DNA in Maternal Serum of Gravid Rhesus Monkeys (Macaca mulatta)

Abstract

The isolation of human fetal DNA from the maternal circulation has provided a source of fetal material for prenatal diagnosis. The objective of this study was to investigate whether a similar pattern could be observed in the maternal circulation of male-bearing gravid rhesus monkeys. A real-time PCR TaqMan system for the rhesus Y-chromosome sex determining region was used to determine fetal sex and to quantify fetal DNA concentrations. Results in 14 healthy pregnancies indicated that fetal male DNA could be routinely detected in maternal serum by 50 d of gestation (late first trimester; term 165 Ϯ 10 d). Fetal DNA concentrations increased with advancing gestation, reaching a mean of 341 genome equivalents/mL of serum (range 11-1570 copies/mL) in the last trimester of gestation, similar to findings in humans. The fetal DNA concentration corresponded to 2.7% of the total maternal serum DNA in the third trimester. Similar to findings in humans, male fetal DNA sequences were not detected postpartum (through 4 wk postpartum) or in animals with a previous history of delivering male offspring. These data indicate that fetal male DNA is present in the maternal circulation of gravid rhesus monkeys comparable to findings in humans and further support the use of this nonhuman primate species as a model to investigate fetomaternal cell trafficking and microchimerism. The discovery of high levels of fetal nucleic acids in the maternal circulation has opened up new areas of investigation and provided a potential new approach for prenatal molecular diagnosis (1). The isolation of cell-free fetal DNA has been shown to be useful material for fetal sex determination and screening for pregnancy-related complications and fetal disease (2, 3). Furthermore, this technique can potentially be applied to investigate other developmental questions associated with the level and degree of fetomaternal trafficking, maintenance of the fetal allograft and tolerance induction, and maternal autoimmune diseases such as scleroderma (4 -14). For exploring such fields of study, appropriate animal models that closely simulate human development and disease are needed.Studies in nonhuman primates, specifically macaques, have shown similarities in anatomy, developmental biology, hematology, and immunology that have prompted the use of this species as a model in fetal and pediatric research (15)(16)(17)(18)(19)(20). Monkeys and humans share many characteristic features because of their close phylogenetic relationship. Developmental similarities include spatial and temporal pattern of organ development, placental structure, and length of gestation. Unlike rodents and sheep, human and nonhuman primates (monkeys) have a similar placental structure, which highlights the importance of this model for addressing questions related to transplacental trafficking of fetal cells and DNA.In this study, we investigated the presence of male fetal rhesus DNA in maternal serum during the length of gestation. In addition, the ⑀-globin gene was used to quantify total (i.e....

Sequential Analysis of α‐ and β‐Globin Gene Expression During Erythropoietic Differentiation from Primate Embryonic Stem Cells

“…The following primers and probes were used for real-time PCR: -globin, forward 5Ј-TGG CAA GGA GTT CAC CCC T-3Ј, reverse 5Ј-AAT GGC GAC AGC AGA CAC C-3Ј, probe 5Ј-FAM-TGC AGG CTG CCT GGC AGA AGC-TAMRA-3Ј; ␥-globin, forward 5Ј-TGG CAA GAA GGT GAC TTC-3Ј, reverse 5Ј-TCA CTC AGC TGG GCA AAG-3Ј, probe 5Ј-FAM-TGG GAG ATG CCA TAA AGA ACC TGG-TAMRA-3Ј; ␤-globin, forward 5Ј-CAA GAA AGT GCT TGG TGC CT-3Ј, reverse 5Ј-GCA AAG GTG CCC TTG AGG T-3Ј, probe 5-FAM-TAG TGA TGG CCT GGC TCA CCT GGA C-TAMRA-3Ј; -globin, forward 5Ј-GGA CCC TCA TTG TGT CCA TGT-3Ј, reverse 5Ј-TGC GGG TAG CTG AGG AAG AG-3Ј, probe 5Ј-FAM-TCC ACT CAG GCC GAC AC-TAMRA-3Ј; ␣-globin, forward 5Ј-TCC CCA CCA CCA AGA CCT AC-3Ј; reverse 5Ј-CCT AAC CTG GGC AGA GCC-3Ј, probe 5Ј-FAM-TCC CCA CTT CGA CCT GAG CCA-TAMRA-3Ј; 18S rRNA, forward 5Ј-AGT CCC TGC CCT TTG TAC ACA-3Ј, reverse 5Ј-GAT CCG AGG GCC TCA CTA AAC-3Ј, probe 5Ј-FAM-CGC CCG TCG CTA CTA CCG ATT GG-TAMRA3Ј. Theand ␤-globin-specific primers and probes have been described previously [26,27]. For all samples, the globin expression levels normalized to the housekeeping gene, 18S rRNA, were determined using the comparative threshold cycle (C T ) method [28,29].…”