[Immortalization of rat epiphysis cartilage cells induced by simian virus 40 large T antigen gene transfection].
OBJECTIVE
To establish immortalized epiphysis cartilage cell strains in order to provide a stable cell resource for cell substitution and gene therapies of growth retardation.


METHODS
Plasmid pEGFP-IRES2-SV40LTag containing simian virus 40 large T antigen gene was transfected into primarily cultured epiphysis cartilage cells of the newborn rat using the lipofectin transfection method. Colonies were isolated by G418 selection and cultured to immortalized cell strains. Fibroblast growth factor receptor-3 (FGFR-3), anti-collagen type II and type X antibodies were used to identify cultured cells and to investigate the capability of differentiation of the transfected cells. SV40LTag expression in expanded cell strains was identified by RT-PCR, Southern blot and immunocytochemistry method.


RESULTS
Anti-G418 cell clone was obtained, which was confirmed as FGFR-3 positive epiphysis cartilage cells with the capability of stable proliferation. mRNA and protein of SV40LTag were expressed in transfected cells after stable transfection. The transfected cells were expanded to immortalized cell strains and named as immortalized epiphysis cartilage cells. The immortalized cells were elliptic or triangular, with two or three short axons. The immortalized epiphysis cartilage cell strains had stable biological characters.


CONCLUSIONS
SV40LTag gene transfection can immortalize epiphysis cartilage cells. The establishment of FGFR-3 positive immortalized epiphysis cartilage cell strains may provide a stable cell resource for cell substitution and gene therapies of growth retardation.
Li
Xin
X
Department of Pediatrics, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China. doctorlixin@hotmail.com
Huang
Shi-Long
SL
Jin
Run-Ming
RM
D000818:Animals; D000952:Antigens, Polyomavirus Transforming / Q000235:genetics*; D002356:Cartilage / Q000166:cytology*; D049109:Cell Proliferation; D004838:Epiphyses / Q000166:cytology*; D007150:Immunohistochemistry; D051381:Rats; D017207:Rats, Sprague-Dawley; D051498:Receptor, Fibroblast Growth Factor, Type 3 / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014162:Transfection
D000952:Antigens, Polyomavirus Transforming; C069820:Fgfr3 protein, rat; D051498:Receptor, Fibroblast Growth Factor, Type 3