Constructing and Cloning of PEGFP-N1 Vector Containing Turncated RPGRIP1L Coiled-Coil Domain from NIH 3T3

Abstract

Ciliopathy is a general term used to describe broad spectrum of diseases caused by primary cilia dysfunction. Different kinds of ciliary gene have been detected to be the underlying causes of this detrimental disease. One of the identified genes is Retinitis Pigmentosa GTPase Regulator 1 Like (RPGRIP1L), which encode transition zone (TZ) protein of the primary cilia. Mutation of this gene has been associated with Joubert Syndrome (JBTS) and Meckel Syndrome (MKS), two of the most lethal ciliopathy affecting multiple organs and system organ, including eyes, kidney, and nervous system. Besides, RPGRIP1L has been shown to play crucial role in proteasomal activity and contribute to primary cilia assembly as its localization will allow proper recruitment of other ciliary proteins. As the first step of gene expression study to elucidate the role of coiled-coil domain on RPGRIP1L localization, this study aims to make a construct containing RPGRIP1L without coiled-coil domain. RNA from NIH 3T3 cells was isolated and used to construct an insert without coiled coil domain. The DNA construct was then inserted to pEGFP-N1 vector and cloned inside Escherichia coli. The colonies were then screened to verify the presence of the insert. Construct of RPGRIP1L without coiled-coil domain was successfully prepared. However, the control for expression study, which was the full RPGRIP1L failed to be ligated to the vector. Further optimization is required to create full RPGRIP1L construct.