Gene Characterization of Hyaluronidase During Embryonic Development of Murine Hearts

Abstract

OBJECTIVES: The objective of this study was to characterize the Hyaluronidase (HYA) gene family throughout gestational development of murine hearts to provide greater insight regarding its role in cardiac morphogenesis. METHODS: Microdissection of murine embryos was accomplished to extract embryonic heart tissue. RNA was extracted using the standard Trizol protocol. cDNA templates were created using a standard protocol. Polymerase chain reaction (PCR) was used to verify presence of HYA, isolate a sample for insertion into a cloning plasmid to make a recombinant clone. A TOPO cloning reaction followed by a double DNA digest was accomplished to verify gene sequencing and orientation in the clone. SYBR Green real time RT- PCR was used to quantify gene expression relative to 18S RNA. RESULTS: RT-PCR provided qualitative data indicating HYA1, HYA2, and HYA3 are present at all observed time points (E8.5, E9.5, E10.5, E11.5, E12.5, E13.5, E14.5, E15.5, and E16.5). Real time RT-PCR data results characterizing relative expression for HYA2: E9.0 (Rel. Exp. = 1.00; SD = 0), E10.5 (Rel. Exp. = 1.33; SD = 0.577), E12.5 (Rel. Exp. = 2.00; SD = 0), E13.5 (Rel. Exp. = 2.66; SD = 0.577), E14.5 (Rel. Exp. = 3.00; SD = 0), E15.0 (Rel. Exp. = 2.00; SD = Error). CONCLUSIONS: HYA1, HYA2, and HYA3 are present at al time points observed in embryonic heart tissue. Relative expression of HYA2 progressively increased from E9.0 until E14.5 and then started tapering downward at time point E15.0.