The Identification Of Amelogenesis Imperfecta-causing Fam83h Mutation And Study Of The Subcellular Localization Of Fam83h

Partâ… The identification of Amelogenesis ImperfectacausingFam83h mutation[Objective] To identify whether the AI phenotypes are caused by Fam83h mutationsin five AI families by DNA sequencing.[Methods] Genomic DNA was isolated from 2 mL of peripheral whole blood of allrecruited members with QIAamp^? DNA Blood Maxi Kit. 7 pairs of primers weredesigned based on human Fam83h cDNA. Full length of Fam83h encoding regions offive probands were first amplified by PCR. All the PCR products were sequenced andthen compared with the original data of Genebank to detect whether there wereFam83h mutations. If there was some Fam83h mutation in the proband, then wewould amplify Fam83h encoding regions of all the recruited members of this familyand send for sequence. If all the affected people had the same Fam83h mutation andall the unaffected people were normal in this site, we could determine that it was thisFam83h mutation that caused AI.[Results] Two new AI-causing Fam83h mutations were found in two families amongfive recruited families. The proband of family 1 was Hispanic. DNA sequencingchromatograms revealed a C to T transition in one of the two FAM83H alleles,changing codon 444 from a glutamine (Q) to a stop (X) codon (g.2452C＞T;c.1330C＞T; p.Q444X), the following 735 amino acids were lost. The proband of family 2 was Caucasian. DNA sequencing chromatograms showed an A to Gtransition in one of the two FAM83H alleles, changing codon 398 from a glutamine(Q) to a stop (X) codon (g.2314C＞T; c.1192C＞T; p.Q398X), the following 781 aminoacids were lost. No Fam83h mutation was found in the other three families[Conclusion] Mutations C＞T at Fam83h cDNA 1330 and 1192 sites lead to AI. Partâ…¡Subcellular localization of Fam83hSectionâ… Construction of Fam83h eukaryotic expression vector[Objective] To construct a Fam83h eukaryotic expression vector fused to greenfluorescence.[Methods] Full-length mouse Fam83h cDNA encoding region was amplified by PCRfrom pBluescriptâ…¡KS+-Fam83h obtained from K.K. DNAFORM (Yokohama, Jpn).Salâ… and Bglâ…¡digestion sites were introduced into the forward and reverse primersrespectively. The PCR product was then inserted into PCR2.1-TOPO vector andtransformed into TOP10. We plated, shaked bacterias and then extracted the PCR2.1-TOPO-Fam83h plasminds. The correct plasmids were first checked by Salâ… / Bglâ…¡double digestion and then sended for sequencing. Fam83h was released from correctPCR2.1-TOPO-Fam83h by Bglâ…¡/Salâ… double digestion and then linked into phrGFPCvector which was digested by BamHâ… /Salâ… . We Transformed Phr-GFP-C-Fam83hinto TOP10 and plated, shaked bacterias and then extracted the phrGFP-C-Fam83hplasminds. The correct plasmids were first checked by Salâ… /Ndeâ… double digestionand then sended for sequencing again.[Results] 3651bp mouse Fam83h encoding region was amplified successfully.Sequence results of PCR2.1-TOPO-Fam83h were coincidence with the genebank data.Sequence results of phrGFP-C-Fam83h showed that Fam83h was inserted into correctregion of phrGFP-C.[Conclusion] We constructed phrGFP-C-Fam83h plasmid successfully, which laidthe first stone for Sectionâ…¡and Sectionâ…¢ Sectionâ…¡Detecting if Fam83h is an intracellular protein[Objective] To determine if Fam83h is an intracellular or secreted protein in vitro.[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×10⁵ cells/cm². 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the cell membrane was stained into red byDiI and nucleus were stained into blue by DAPI. The green fluorescence fromFam83h was detected in fluorescence microscope and then taken pictures.[Results] We detected green fluorescence of Fam83h was always within the redmembrane in HEK293 cell lines, which suggested definitely that Fam83h should bean intracellular protein instead of being secreted extracellularly. Further more, wefound that green fluorescence of Fam83h seldom overlapped with nucleus, most ofthem located in perinuclear region, especially the front edge of membraneinvagination, where usually contains Golgi apparatus. Regard the inaccuracy causedby angle of taking pictures, we propose that Fam83h probably locates in cell organsaround nucleus, especially Golgi apparatus[Conclusion] Fam83h is an intracellular protein, but not in nucleus. The subcellularlocalization of Fam83h is probably in cell organs around nucleus, it is speculatedclosely related with Golgi complex. Sectionâ…¢Study of the subcellular localization of Fam83h[Objective] To determine the relation between the subcellular localization of Fam83hand Golgi complex.[Methods] HEK293 cells were refreshed and then seeded into chamber slides whenthey were in good condition, with seeding density 2-6×10⁵ cells/cm². 24 hrs later, thecorrect phrGFP-Fam83h plasmid was transfected into HEK293 cell lines by use ofLipofectamine 2000. The ratio of phrGFP-Fam83h: Lipofectamine 2000 was 1μg: 1μl.Cell culture media was changed for normal media after 6 hrs. 24-72 hrs later aftertransfection cells were fixed by 4% PFA, the nucleus were stained into blue by DAPIand Golgi complex was stained into red by BODIPY. The relation between greenfluorescence from Fam83h and red fluorescence from Golgi complex was detected influorescence microscope and then taken pictures.[Results] In HEK293 cells, green fluorescence signal from Fam83h was always inand smaller than the red fluorescence from Golgi complex, which suggested stronglythat Fam83h shoul be in Golgi complex. Typically the Golgi stain was weaker ornegative where the GFP localized, it seemed that Fam83h located in the membrane ofGolgi complex.[Conclusion] The subcellular localization of Fam83h is closely related with Golgicomplex, Fam83h may locate in the membrane of Golgi complex.