Mentor: Jennifer Schumacher, Ph.D.

Congenital heart disease (CHD) is highly prevalent in live births and requires concise diagnoses and treatment plans [1]. Although there is a vast amount of cardiovascular genetics research in the field of CHD, there are still gaps in knowledge regarding the gene networks and cellular processes that drive heart development and function. Although many cardiac genes have been studied, there are still open questions surrounding the gene networks that regulate specific stages of heart development. Six genes with little to no previous expression data in the heart were selected for further molecular analysis. A previous single-cell RNA sequence showed that these genes were co-expressed with myh7 in the ventricle at 96 hours post fertilization (hpf). Forward and reverse primers were designed for each gene. To create a complementary DNA (cDNA) library, the total RNA was extracted from Danio Rerio (zebrafish) at 96 hpf. Using reverse transcription, mRNA was isolated and transcribed into cDNA. To amplify cDNA through polymerase chain reaction (PCR), forward and reverse gene-specific primers were used. Once we confirmed a successful PCR product via gel electrophoresis, we used bacterial transformation to clone the plasmid DNA. In order to isolate the plasmid DNA, minipreps will be used. We will then confirm isolation using a restriction digest and gel electrophoresis. Moving forward with this experiment, we will create RNA probes to conduct in-situ hybridization (ISH). When analyzing the temporal and spatial expression throughout heart development using ISH, we can draw conclusions about each gene’s potential functions during heart development.

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