Electroporation of Embryonic Kidney Explants
Metanephric kidney development in the mouse begins at embryonic day (E) 10.5, when the ureteric bud (UB), an outgrowth of the epithelial nephric duct, invades the neighboring metanephric mesenchyme (MM). The ureteric bud then undergoes a series of branching events to form the collecting duct network of the adult kidney (Fig. 19.1). As each ureteric bud tip forms, the adjacent undifferentiated mesenchyme is induced to epithelialize and form a nephron, the functional unit of the adult kidney that filters waste. Rodent embryonic kidneys can be dissected and cultured as explants such that branching morphogenesis and nephrogenesis can be observed ex vivo (Rothenpieler and Dressler, 1993; Vega et al., 1996; Piscione et al., 1997; Gupta et al., 2003).
The roles of signaling molecules and transcription factors during kidney development have been studied in explant cultures by introducing small interfering RNA (Davies et al., 2004), by treating with pharmaceutical agents (Tang et al., 2002), or by using viral transduction to overexpress mutant proteins (Favre et al., 2000). More recently, microinjection followed by electroporation has been used to successfully express plasmid DNA encoding proteins important for ureteric bud induction, branching morphogenesis, and nephrogenesis in the developing kidney (Gao et al., 2005; Self et al., 2006; Alie et al., 2007). This approach allows for both gain-of-function and loss-of-function experiments in a high-throughput, cost-effective manner. Tissue-specific effects can also be analyzed: DNA constructs can be delivered to either the metanephric mesenchyme or the ureteric bud lineages to evaluate the primary effects of genetic perturbation within a target cell population. This method can be used to evaluate the function of any gene of interest to determine whether it is then feasible to invest in the creation of more labour-intensive conditional knock-out or knock-in mouse lines. From expression profiling technologies such as microarrays and SAGE, a large number of genes have been implicated in kidney development (Meyer et al., 2004; Schmidt-Ott et al., 2005), yet for most of these genes, their function in organogenesis remains uncharacterized. Microinjection with electroporation therefore provides an economical means to rapidly evaluate the function of previously described genes, and to characterize the role of genes more recently implicated in kidney development.
KeywordsPulse Time Kidney Development Enhance Green Fluorescent Protein Expression Metanephric Mesenchyme Nephric Duct
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