Establishment of ameloblast cell strain controlled by the pTet-on regulatory systerm

LI Dong-liang; LIANG Guang-zhi; HE Yong-yun; LIU Xiao-ying; SUN Yan; ZHANG Juan-juan; LI Wu-xiu; GAO Yu-guang

Abstract：AIM:To establish an eukaryotic expression system of ALC-pTet-on advanced cell strain which could be induced by doxycycline to highly express foreign genes for gene function studies.METHODS:The ALCs were transfected with pTet-on advanced vector by using TransFast~(TM) transfection reagent.and the anti-clones were chosen by geneticin(G418)selecting.The stably transfected cells could survive in neomycin-resistant culture medium containing1000μg/ML G418.The expression level of pTet-on Advanced gene was examined by reverse transcription-polymerase chain reaction(RT-PCR).The function of the positive cell clones was screened using luciferase reporting gene assay kit.RESULTS:Totally 13 Tet-on Advanced positive ALC cell clones were obtained after G418 screening.The RT-PCR results showed that all clones expressed Reverse Tet-Controlled Transactivator(rtTA Advanced)and the sequencing was matched with the pTet-on Advanced gene.It indicated that pTet-on Advanced gene had been integrated into the genomic DNA of ALC cells.One strain of pTet-on positive ALC cell clones with multiple inducing expression rate of 22.1 was obtained by luciferase reporting gene assay.There was no difference in the morphology and growth rate between pTet-on Advanced positive ALCs and normal ALC cells.CONCLUSION:One pTet-on Advanced positive ALC cell strain with multiple inducing expression rate of 22.1 was established successfully,which could be a base for the function studies of EMPs in ameloblasts.