Adenoviral-mediated gene delivery to trophoblast cells.
ABSTRACT This chapter focuses on technology for construction of recombinant adenoviruses containing reporter genes under the control of putative regulatory regions of the human (h)CYP19 (aromatase) gene, as well as expression vectors. These recombinant adenoviruses have been used in primary cultures of human placental cells to characterize regulatory regions of the hCYP19 gene and to analyze the function of transcription factors on hCYP19 expression and on trophoblast differentiation.
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ABSTRACT: Studies in primary human trophoblasts provide critical insights into placental function in normal and complicated pregnancies. Mechanistic studies in these cells require experimental tools to modulate gene expression. Lipid-based methods to transfect primary trophoblasts are fairly simple to use and allow for the efficient delivery of nucleic acids, but potential toxic effects limit these methods. Viral vectors are versatile transfection tools of native trophoblastic or foreign cDNAs, providing high transfection efficiency, low toxicity and stable DNA integration into the trophoblast genome. RNA interference (RNAi), using small interfering RNA (siRNA) or microRNA, constitutes a powerful approach to silence trophoblast genes. However, off-target effects, such as regulation of unintended complementary transcripts, inflammatory responses and saturation of the endogenous RNAi machinery, are significant concerns. Strategies to minimize off-target effects include using multiple individual siRNAs, elimination of pro-inflammatory sequences in the siRNA construct and chemical modification of a nucleotide in the guide strand or of the ribose moiety. Tools for efficient gene targeting in primary human trophoblasts are currently available, albeit not yet extensively validated. These methods are critical for exploring the function of human trophoblast genes and may provide a foundation for the future application of gene therapy that targets placental trophoblasts.Placenta 07/2012; 33(10):754-62. DOI:10.1016/j.placenta.2012.07.003 · 3.29 Impact Factor
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ABSTRACT: Mononuclear cytotrophoblasts of the human placenta proliferate rapidly, subsequently fuse and differentiate to form multinucleated syncytiotrophoblast with induction in aromatase/hCYP19A1 and chorionic gonadotropin (hCGβ) expression. Using microarray analysis, we identified members of the miR-17∼92 cluster and its paralogs, miR-106a∼363 and miR-106b∼25, that are significantly downregulated upon syncytiotrophoblast differentiation. Interestingly, miR-19b and miR-106a directly targeted hCYP19A1 expression, while miR-19b also targeted hGCM1, a transcription factor critical for mouse labyrinthine trophoblast development. Overexpression of these miRNAs impaired syncytiotrophoblast differentiation. hGCM1 knockdown decreased hCYP19A1 and hCGβ expression, substantiating its important role in human trophoblast differentiation. Expression of the protooncogene, c-Myc, was increased in proliferating cytotrophoblasts, compared to differentiated syncytiotrophoblast. Moreover, c-Myc overexpression upregulated miR-17∼92 and inhibited hCYP19A1 and hCGβ expression. Binding of endogenous c-Myc to genomic regions upstream of the miR-17∼92 and miR-106a∼363 clusters in cytotrophoblasts dramatically decreased upon syncytiotrophoblast differentiation. Intriguingly, we observed higher levels of miR-106a and -19b and lower aromatase and hGCM1 expression in placentas from preeclamptic women, as compared to placentas from gestation-matched normotensive women. Our findings reveal that c-Myc-regulated members of miR-17∼92 and miR-106a∼363 clusters inhibit trophoblast differentiation by repressing hGCM1 and hCYP19A1 and suggest that aberrant regulation of these miRNAs may contribute to the pathogenesis of preeclampsia.Molecular and Cellular Biology 02/2013; DOI:10.1128/MCB.01228-12 · 5.04 Impact Factor