Transient, Inducible, Placenta-Specific Gene Expression in Mice

Abstract

Molecular understanding of placental functions and pregnancy disorders is limited by the absence of methods for placenta-specific gene manipulation. Although persistent placenta-specific gene expression has been achieved by lentivirus-based gene delivery methods, developmentally and physiologically important placental genes have highly stage-specific functions, requiring controllable, transient expression systems for functional analysis. Here, we describe an inducible, placenta-specific gene expression system that enables high-level, transient transgene expression and monitoring of gene expression by live bioluminescence imaging in mouse placenta at different stages of pregnancy. We used the third generation tetracycline-responsive tranactivator protein Tet-On 3G, with 10- to 100-fold increased sensitivity to doxycycline (Dox) compared with previous versions, enabling unusually sensitive on-off control of gene expression in vivo. Transgenic mice expressing Tet-On 3G were created using a new integrase-based, site-specific approach, yielding high-level transgene expression driven by a ubiquitous promoter. Blastocysts from these mice were transduced with the Tet-On 3G-response element promoter-driving firefly luciferase using lentivirus-mediated placenta-specific gene delivery and transferred into wild-type pseudopregnant recipients for placenta-specific, Dox-inducible gene expression. Systemic Dox administration at various time points during pregnancy led to transient, placenta-specific firefly luciferase expression as early as d 5 of pregnancy in a Dox dose-dependent manner. This system enables, for the first time, reliable pregnancy stage-specific induction of gene expression in the placenta and live monitoring of gene expression during pregnancy. It will be widely applicable to studies of both placental development and pregnancy, and the site-specific Tet-On G3 mouse will be valuable for studies in a broad range of tissues.

Endometrial VEGF induces placental sFLT1 and leads to pregnancy complications

“…These were performed using procedures, probes, primers, and antibodies described previously by us and others (27,35,45,47). See Supplemental Methods for details.…”

“…Lentivirus construction and production were described previously (27,46,47). Briefly, LV-Fluc/GFP was prepared by incorporating the coding sequences for firefly luciferase (Fluc) and EGFP linked by an encephalomyocarditis virus internal ribosomal entry site (IRES) sequence (27,46).…”

“…The CMV promoter was removed for construction of LV-H1-sFLT1shRNA-EF1α-copGFP following a standard cloning protocol. Viral particles were produced using standard calcium phosphate transfection in human embryonic kidney fibroblasts (293FT), concentrated using PEG-it Virus Precipitation Solution (System Biosciences), resuspended in PBS, aliquoted, and stored at -80°C (27,47). Virus titer (particles/ml) was determined using the QuickTiter Lentivirus Quantitation Kit (Cell Biolabs Inc.) as described previously (27,47).…”

“…sFlt1 expression in the placenta was knocked down using lentivirus-mediated, placenta-specific expression of sFLT1 shRNA, as described previously (27,47). Blastocysts were transduced with either control (LV-copGFP) or sFLT1 shRNA (LV-sFLT1shRNA-copGFP).…”

“…Blastocysts were transduced with either control (LV-copGFP) or sFLT1 shRNA (LV-sFLT1shRNA-copGFP). Optimal virus concentration, incubation time, selection of optimally transduced blastocysts, and the procedure for blastocyst transfer into pseudopregnant recipients (n = 18) have been described previously (27,47). Maternal blood, urine, and tissue samples from various organs (including kidneys, livers, lungs, brains, deciduas, placentas, and fetuses) were collected on…”