Constructing and Cloning of Pegfp-N1 Vector Containing Turncated RPGRIP1L C2 Domain from NIH3T3

Abstract

Primary cilia is an antenna like organelle that functions as signal transmitter in eukaryotic cells. RPGRIP1L is one of the protein that is located on the primary cilia, which plays an important role in controlling cellular signaling, assembling the ciliary base, and controlling autophagy. RPGRIP1L is composed of 3 main domains, namely Coiled-coil, C2, and RIDL domain. Among those domains of RPGRIP1L, C2 domain is suspected to act as an anchor of the RPGRIP1L to the transition zone of the primary cilia. By knocking down the C2 domain in RPGRIP1L gene, RPGRIP1L protein with truncated C2 domain can be later produced in mammalian cells and the C2 domain role can be subsequently observed. The construction of pEGFP-N1 containing RPGRIP1L with truncated C2 domain was carried out through combining the pEGFP-N1 with Coiled-coil domain insert of RPGRIP1L, followed by final cloning of pEGFP-N1 vector containing Coiled-coil domain with RIDL domain of RPGRIP1L. Another clone of pEGFP-N1 containing the full sequence of RPGRIP1L was also generated as the experiment control. However, the desired construct containing the truncated C2 domain of RPGRIP1L and control construct containing the full sequence of RPGRIP1L was not achieved. This might be caused by incomplete digestion, inefficient ligation, and too low concentration of digested product during the cloning process. Therefore, further experiments with more optimum cloning condition needs to be done to generate the clone containing RPGRIP1L with the truncated C2 domain and full sequence of RPGRIP1L.