Rpgrip1l Knockout NIH 3T3 Cell Line

The Rpgrip1l Knockout NIH 3T3 Cell Line is a CRISPR/Cas9-edited knockout cell line designed for targeted disruption of the mouse Rpgrip1l gene. This product provides a stable loss-of-function model in a well-characterized fibroblast background, enabling rigorous investigation of Rpgrip1l-dependent cellular processes. The knockout cell line has been engineered using CRISPR/Cas9-mediated genome editing to eliminate functional Rpgrip1l protein expression, without introducing specific off-target modifications. The resulting line offers a consistent and reproducible system for studying primary cilium formation and associated signaling pathways.

NIH 3T3 cells are a spontaneously immortalized mouse embryonic fibroblast line known for contact-inhibited growth and robust primary cilium assembly upon serum starvation. These cells are widely employed in cell biology research due to their well-defined signaling networks, particularly the Hedgehog and Wnt pathways. Their fibroblast origin and stable karyotype make them an ideal platform for examining ciliary biology and centrosome-related functions.

Rpgrip1l encodes a centrosomal and basal body protein that is a critical component of the ciliary transition zone, where it maintains the barrier function essential for proper ciliary protein trafficking. The protein interacts with key ciliopathy-associated factors such as RPGR, NPHP1, and CEP290 to regulate transition zone integrity. This function is required for primary cilium formation and for Hedgehog signal transduction, as Rpgrip1l facilitates the localization of pathway components including SMO, SUFU, and GLI transcription factors within the cilium. Rpgrip1l expression is transcriptionally regulated by RFX3 and FOXJ1, and it functions downstream of SHH to modulate the activity of target genes GLI1, GLI2, and GLI3. Loss of Rpgrip1l disrupts ciliary protein entry, impairing GLI activation and altering downstream effectors such as CCND1 and BCL2.

In the NIH 3T3 context, deletion of Rpgrip1l results in defective ciliogenesis and compromised Hedgehog pathway responsiveness. Because these cells normally assemble primary cilia and engage Hedgehog signaling during cell cycle arrest, the knockout line provides a physiologically relevant model for dissecting the molecular requirements for cilium-dependent signal transduction. The model is particularly valuable for studying how transition zone components influence the trafficking of ciliary receptors and downstream effectors, and for elucidating the mechanistic links between ciliary dysfunction and developmental disorders such as Joubert syndrome, Meckel syndrome, and nephronophthisis.

This knockout cell line supports diverse applications, including ciliopathy modeling, screening for ciliogenesis modulators, and Hedgehog pathway analysis. Typical assays include immunofluorescence for ciliary markers ARL13B and acetylated ??-tubulin, Western blot for GLI1/GLI3, and RT-qPCR for Hedgehog targets. Co-immunoprecipitation and cell cycle analysis enable study of transition zone protein interactions and proliferative consequences of cilium loss. This line is an essential tool for ciliopathy research and therapeutic development. For further details, please contact Ascent Research.