Abstract
The delivery of an exogenous or modified DNA into the mitochondria of mammalian cells has been proposed as an alternative way to prevent or modify mitochondrial genetic defect. In  this study, a lipofection method was developed to transfer exogenous DNA into mitochondria of BRIN BD11 cells, a rat beta pancreas cell line, and human myoblast primary cells. An artificial mini-mitochondrial genome carrying a reporter gene encodes for a mitochondrial green fluorescent protein (mtGFP) was usedto track the fate of the exogenous DNA following its transfection into cellular mitochondria. Epifluorescence microscopic observation was carried out to detect the green fluorescence resulted from the GFPexpression. Immunohistochemical assays were also performed to validate and confirm the GFP expression. Collectively, the results indicate that the exogenous DNA pmtGFP could be  delivered into the mitochondrial compartment and was functional withinthe mitochondria of BRIN BD11 and human myoblast cells. Such achievement is a significant progress as both the artificial DNA construct and the mitochondrial transfection method could serve as a model to study the dynamic of mutant mtDNA as well as to provide a basis for the development of gene therapy for human mitochondrial diseases. Key words: DQAsome, pmtGFP, beta pancreas cell line, human myoblast primary cells
Highlights
The delivery of an exogenous or modified DNA into the mitochondria of mammalian cells has been proposed as an alternative way to prevent or modify mitochondrial genetic defect
An artificial mini-mitochondrial genome carrying a reporter gene encodes for a mitochondrial green fluorescent protein was used to track the fate of the exogenous DNA following its transfection into cellular mitochondria
Epifluorescence microscopic observation was carried out to detect the green fluorescence resulted from the green fluorescent protein (GFP) expression
Summary
Sel beta pankreas yang digunakan adalah sel BRIN BD11 passage 44 (pemberian Dr Aris Wibudi, Institut Pertanian Bogor, Bogor, Indonesia). Sel BRIN BD 11 dikultur pada slide kultur 4 ruang (Chamber Slide Culture Chambers Permanox, Cat. No 1774371, Nunc) atau pada coverslips dalam 35 mm culture dishes (Nunc) dengan media kultur RPMI 1640 yang mengandung 10% fetal bovine serum (FBS, Gibco), 100 IU/mL Penicillin (Gibco), 100 μg/mL Streptomycin (Gibco) dan 1 mM L-glutamine (Gibco). Sel yang digunakan untuk transfeksi dikultur pada slide kultur 4 ruang atau pada coverslips dalam 35 mm culture dishes dengan media kultur HAM F-12 (Gibco, Invitrogen) yang mengandung 20% fetal bovine serum, 100 IU/mL Penicillin, 100 μg/mL Streptomycin dan 1 mM L-glutamine. Konstruk DNA yang digunakan untuk transfeksi mitokondria sel BRIN BD11 dan myoblast manusia adalah pmtGFP, sedangkan liposom yang digunakan adalah DQAsome. Larutan kemudian disentrifugasi pada 3000 rpm selama 30 menit dan supernatan yang diperoleh digunakan sebagai liposome untuk transfeksi. Perlakuan yang sama diberikan kepada sel myoblast manusia, hanya saja lipopleks diganti DQAsome saja atau media kultur
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