Ex vivo Trophoblast-specific Genetic Manipulation Using Lentiviral Delivery

Background

The placenta functions as a conduit between the mother and the developing fetus and is essential for viviparity (Georgiades et al., 2002). It is specialized to facilitate and coordinate maternal adaptations to pregnancy and fetal development (Soares et al., 2014; Burton et al., 2016). The placenta contains trophoblast cells, which perform several specialized functions. Acquisition of trophoblast cell specializations requires highly regulated differentiation of trophoblast stem and progenitor cell populations (Maltepe and Fisher, 2015). The mature rat placenta is comprised of two morphologically and functionally distinct compartments: the junctional zone and the labyrinth zone (Soares et al., 2012). Progenitor cells, spongiotrophoblast cells, glycogen trophoblast cells, and polyploid trophoblast giant cells comprise the junctional zone. An invasive trophoblast lineage arises from progenitors within the junctional zone region (Ain et al., 2003; Soares et al., 2014). During the last week of gestation, these cells move out of the placenta and invade into the maternal uterine mesometrial compartment (Ain et al., 2003; Pijnenborg and Vercruysse, 2010). The innermost layer of the placenta (proximal to the developing fetus) is called the labyrinth zone, which consists of trophoblast cells and fetal vasculature derived from the allantois. Progenitor trophoblast cells within the labyrinth zone fuse to form syncytia, which provide barriers between maternal and fetal compartments (Soares et al., 2012). The labyrinth zone consists of an elaborate branched structure, providing a large surface area for nutrient, waste and gas exchange with the fetus (Knipp et al., 1999; Watson and Cross, 2005).

Specific modifications of the trophoblast lineage can be achieved using lentiviral transduction of the outer layer of the embryo at the blastocyst stage, termed trophectoderm (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009) (Figure 1). This method allows for efficient manipulation of all trophoblast cell lineages. It also facilitates discrimination between trophoblast and embryonic contributions to phenotypic outcomes arising from global genetic manipulations. The experimental basis for selective trophoblast viral infection is directly related to the structure of the blastocyst and the tight epithelium formed by the outer trophectoderm, which effectively restricts viral particle access to the inner cell mass (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009). This method can be utilized to investigate the consequences of both ‘gain-of-function’ and ‘loss-of-function’ manipulations of the trophoblast lineage, which can provide insights into molecular mechanisms controlling placental development (Georgiades et al., 2007; Malashicheva et al., 2007; Okada et al., 2007; Lee et al., 2009; Morioka et al., 2009; Kent et al., 2011; Chakraborty et al., 2016; Muto et al., 2016).


Figure 1. Schematic showing experimental plan for lentiviral transduction of blastocysts and downstream analyses. Blastocysts are transduced with lentiviral particles (shown in green) expressing specific ‘gain-of-function’ or ‘loss-of-function’ constructs and cultured for 72 h for outgrowth assays or directly transferred to day 3.5 pseudopregnant animals for in vivo analyses.