Figure 1.
Diagrammatic representation of the lentivirus vector construct and trophoblast-specific lentiviral gene delivery.
A, the lentiviral double-fusion reporter gene construct (LV-Fluc/Tomato). Fluc/Tomato was cloned downstream of the ubiquitin C (Ubi C) promoter with a 14-amino acid (LENSHASAGYQAST) linker. B, zona-free blastocysts were transduced with LV-Fluc/Tomato and transduction efficiency of each blastocyst was evaluated by BLI and Tomato fluorescence. Optimally transduced blastocysts were then transferred into pseudopregnant recipients. Fluc expression in the placenta was assessed by BLI at various stages of gestation following intraperitoneal injection of D-luciferin, and Tomato expression in different cell types was assessed after collection of placentas on GD18. Note that these strategies permit quantitative assessment of placenta-specific transgene expression in the same animal at different stages of pregnancy.
Figure 2.
Trophoblast-specific Fluc expression assessed by live BLI at different stages of pregnancy.
Blastocysts (selected) optimally transduced with LV-Fluc/Tomato were transferred into GD3 pseudopregnant recipients and Fluc expression in the placenta was evaluated by BLI at different stages of pregnancy in the same animal. A–E, grayscale body surface images and pseudocolor luminescence images (blue - least intense, red - most intense) were superimposed; photons emitted from implanting blastocysts could be detected as early as GD5 (A). F, placenta-specific Fluc (BLI) and Tomato (fluorescence) expression on GD18. Note that fetuses of the corresponding placentas are both Fluc and Tomato negative, indicating viral transduction of trophoblast-specific lineage. G, levels of total photon flux over the abdominal area at different stages of pregnancy; there was an exponential increase in signal intensity from GD6 through GD12.
Figure 3.
Trophoblast-specific Tomato expression by lentivirus-mediated transgene delivery into blastocysts.
Tomato expression was examined in zona-free blastocysts infected with LV-Fluc/Tomato (A–B) and in GD18 placentas (C–F) after blastocyst transfer into pseudopregnant mice. A–B, phase-contrast (A) and fluorescence (B) images showing Tomato expression in the trophectoderm of blastocysts (A, B, ▾) and not in the inner cell mass (A, B, *). C–F, Tomato expression in trophoblast lineages of placentas; D–F, magnified areas in C; giant cells (GC), spongiotrophoblast (Sp) and labyrinth (La) layers, decidua (De), myometrium (My), and chorionic plate (CP).
Table 1.
Effect of lentivirus transduction (LV-Fluc/Tomato) into blastocysts and live bioluminescence imaging on pregnancy outcome.
Figure 4.
Wide variability in Fluc expression among placentas of the same litter despite identical conditions of viral transduction of blastocysts.
A–D, BLI of Fluc expression in placentas at different stages of pregnancy; E, dramatic variations in Fluc expression among different placentas from the same litter collected on GD18; F, detection of very weak Fluc signal in a placenta (marked in E) after lowering the threshold of BLI. G, Tomato florescence images of the placentas shown in E.
Figure 5.
Selection of blastocysts for optimal lentivirus transfection efficiency.
Each LV-Fluc/Tomato transduced blastocyst was incubated in KSOM containing D-luciferin (50 ug/ml) and assessed for Fluc expression (luciferase activity) by BLI. A, wide variations in Fluc signals from different blastocysts despite identical virus transfection conditions; B–C, blastocysts with BLI values between 2.0E+4 and 6.0E+4 p/s/cm2/sr were selected for transfer. Increase of BLI threshold to 2.0E+4 p/s/cm2/sr identifies blastocysts with low signals (B, *), and further increase in threshold to 6.0E+4 p/s/cm2/sr identifies blasocysts with very high intensity signals (C, ▾).