Authors:

Haley Strauch

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Introduction: Congenital heart defects (CHD) are the most common birth defect in the United States. Pregnancies complicated with CHD have been linked to improper development/function of the placenta. NOTCH1 mutations are more common in CHD patients than in the general population. This study aims to optimize a protocol for a NOTCH1 knockdown using siRNA in BeWo choriocarcinoma cells as a model of cytotrophoblast and evaluate the role that NOTCH1 plays in placental development. Methods: BeWo cells were maintained in F-12 Ham Complete Growth media tat 37°C and 5% CO2. BeWo cells were plated at 200,000 cells per well of a 6- well plate and cultured overnight. Media was changed to minimal media and Lipofectamine optimization performed: increasing volumes from 2ul of lipofectamine were complexed with 2ul of NOTCH1 or scramble siRNA and added to cells. Six hours after transfection, fetal bovine serum (FBS) was added to the cells. 48 hours later cells underwent either crystal violet assay to assess cell proliferation or RNA extraction using the Qiagen RNeasy Plus Mini Kit. Following RNA extraction and cDNA conversion qPCR was performed to evaluate NOTCH1 knockdown. Results: BeWo proliferation was seen best at a concentration of 2ul of Lipofectamine compared to 4ul of Lipofectamine. In the NOTCH1 siRNA KD cells there was a 41% percent reduction in NOTCH1 expression compared to cells treated with scramble siRNA. Discussion: NOTCH1 is a crucial pathway during early pregnancy that impacts trophoblast cells and transport pathways in the placenta. Optimizing the knockdown of NOTCH1 helps develop a model of dysfunctional placentas, which can be used for future experiments investigating specific mechanisms of disrupted NOTCH1 in cytotrophoblast differentiation. Optimization for the NOTCH1 Knockdown in

Source:

University of Florida / Haley Strauch, Alyssa Tipler, Helen Jones / 2023

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Co-authors:

Haley Strauch