Important aspects of placental-specific gene transfer

Abstract

The placenta is a unique and highly complex organ that develops only during pregnancy and is essential for growth and survival of the developing fetus. The placenta provides the vital exchange of gases and wastes, the necessary nutrients for fetal development, acts as immune barrier that protects against maternal rejection, and produces numerous hormones and growth factors that promote fetal maturity to regulate pregnancy until parturition. Abnormal placental development is a major underlying cause of Pregnancy-Associated Disorders that often result in preterm birth. Defects in placental stem cell propagation, growth, and differentiation are major factors that affect embryonic and fetal well being and dramatically increase the risk of pregnancy complications. Understanding the processes that regulate placentation is important in determining the underlying factors behind abnormal placental development. The ability to manipulate genes in a placenta-specific manner provides a unique tool to analyze development and eliminates potentially confounding results that can occur with traditional gene knockouts. Trophoblast stem cells and mouse embryos are not overly amenable to traditional gene transfer techniques. Most viral vectors, however; have a low infection rate and often lead to mosaic transgenesis. While the traditional method of embryo transfer is intrauterine surgical implantation, the methodology reported here, combining lentiviral blastocyst infection and nonsurgical embryo transfer, leads to highly efficient and placental-specific gene transfer. Numerous advantages of our optimized procedures include increased investigator safety, a reduction in animal stress, rapid and noninvasive embryo transfer, and higher a rate of pregnancy and live birth.

Trophoblast-Specific Expression of Hif-1α Results in Preeclampsia-Like Symptoms and Fetal Growth Restriction

“…This lentivirally-driven gene delivery results in expression of the target gene exclusively in trophoblast cells of the placenta, as indicated by V5 immunohistochemistry, without altering maternal or embryonic tissue (Fig. S1) 38 . To investigate the effects of prolonged CA-Hif-1α, we confirmed the stability of Hif-1α protein in normoxia by Western blot (Figs 1a and S2), while functional activation of a direct and Hif-1α-specific target was confirmed using Pgk1-luciferase (Fig.…”

“…The lentiviral construct pLB2V5 was generated by replacing the green fluorescent protein (GFP) gene of pLv-CMV-[GFP]-V5 with the multiple cloning site of pBSSK+ 38 . pLB2V5-[CA-Hif-1α] was cloned by ligating the mouse Hif-1 α triple site-directed mutant cDNA from pc3-Hif-1α3XSDM into the pLB2V5 vector by restriction enzyme digest and confirmed by sequencing (Cleveland Genomics, Cleveland, OH) 34 .…”

“…C57BL/6 females were superovulated and mated as previously described 38 . Copulation plugs found the following morning were designated as E0.5.…”

“…Copulation plugs found the following morning were designated as E0.5. Superovulated C57BL/6 female embryos were collected at E1.5 (2-cell) in M2 media and cultured in pre-equilibrated 20 µl KSOM AA microdrops at 37.5 °C and 5.5% CO2 until E3.5 blastocysts 38 . ICR females were mated with vasectomized ICR males to generate pseudopregnancy, checked for copulation plug the next morning and designated as 0.5 d.p.c.…”

Development and Validation of a Rex1-RFP Potency Activity Reporter Assay That Quantifies Stress-Forced Potency Loss in Mouse Embryonic Stem Cells

“…To produce transgenic reporter mESCs without the stress of electroporation or cationic lipids, lentivirus infection of parental ESCs is used [21]. Instead of antibiotic selection, transgenic ESCs are passaged in a normal manner, but fluorescence-activated cell sorting (FACS) is used to select stem cells reporting pluripotency.…”