Establishment and phenotypic identification of an immortalized mouse corneal epithelial cell line

Abstract

Objective To investigate the method for establishing an immortalized mouse corneal epithelial cell line, and to identify the stability of its phenotype. Methods Primary mouse corneal epithelial cells were extracted and transfected with Simian Vacuolating Virus 40 Large T Antigen (SV40-LT) lentivirus to establish an immortalized cell line. HE staining and immunofluorescent staining were used to identify the phenotypes and markers (K12, Pax6, Ki-67) of corneal epithelium. Primary cor-neal epithelial cells were extracted to identify K14 protein in corneal epithelial cell layer, and bright field observation was performed to evaluate cell morphological characteristics. Several markers (K12, Pax6, Ki-67, ABCG2, K14) were identified for P1 and P8 cells after transfection. Crystal violet staining was used to observe the colony-formation ability of P1 and P8 cells after transfection. Results Mouse corneal epithelium was stained positive for K12, Pax6 and Ki-67, while the P1 and P8 generations of mouse cor-neal epithelial cells were stained positive for K12, Pax6, Ki-67, K14, and ABCG2; there were no significant differences in the positive rates of the above markers (P>0.05). There was no significant difference in colony-formation ability between P1 and P8 cells (P>0.05). Conclusion The immortalized mouse corneal epithelial cell line is successfully established by transfection with SV40-LT lentivirus, which has stable phenotype and proliferation.

Keywords

• cell line|epithelial cells|cornea|transfection|genetic markers|mice, inbred c57bl