Subject(s)
Bone Regeneration/physiology , Bone Substitutes/therapeutic use , Polymers/therapeutic use , Tissue Engineering/methods , Tissue Scaffolds/trends , Animals , Biocompatible Materials/standards , Biocompatible Materials/therapeutic use , Bone Diseases/therapy , Bone Substitutes/standards , Humans , Intercellular Signaling Peptides and Proteins/pharmacology , Intercellular Signaling Peptides and Proteins/therapeutic use , Materials Testing/methods , Materials Testing/standards , Osteogenesis/physiology , Polymers/standards , Stem Cell Transplantation/methods , Stem Cell Transplantation/trends , Stem Cells/physiology , Tissue Engineering/trends , Tissue Scaffolds/standardsABSTRACT
Human marrow stromal cells (hMSCs) are an attractive source of adult stem cells for autologous cell and gene therapy. To transfect hMSCs without the use of viruses, we developed improved conditions for stable transfection of the cells by electroporation. hMSCs were isolated by adherence to plastic, and were electroporated at 600 V and 100 micros in a 2-mm gap cuvette with a plasmid containing enhanced green fluorescence protein (EGFP) and neomycin phosphotransferase gene (neo(r)). After electroporation of 10(6) cells with 10 microg of the linearized plasmid DNA, hMSCs with stable DNA integration were selected by culturing with 200 microg/ml G418. The transfected hMSCs were expanded another 300-fold in 14 days to obtain 89 million cells, of which 98% expressed EGFP. Chloroquine increased the number of hMSCs transiently expressing EGFP from 12% to over 50%, but decreased stable integration. Stable integration of plasmid DNA into rat MSCs by electroporation was also successful. The transfected MSCs retained their capacity to differentiate into both adipocytes and osteoblasts. Thus, MSCs were stably transfected with plasmid DNA and retained their differentiation capacity after expansion.
Subject(s)
Bone Marrow Cells , DNA/administration & dosage , Electroporation/methods , Genetic Therapy/methods , Transfection/methods , Adipocytes/cytology , Animals , Bone Marrow Cells/cytology , Cell Differentiation , Cell Division , Cell Line , Chloroquine/pharmacology , Flow Cytometry , Gene Expression , Green Fluorescent Proteins , Humans , Luminescent Proteins/genetics , Male , Osteoblasts/cytology , Rats , Rats, Inbred Lew , TransgenesABSTRACT
Expanded versions of the Ribosomal RNA Mutation Databases provide lists of mutated positions in 16S and 16S-like ribosomal RNA (16SMDBexp) and 23S and 23S-like ribosomal RNA (23SMDBexp) and the identity of each alteration. Alterations from organisms other than Escherichia coli are reported at positions according to the E.coli numbering system. Information provided for each mutation includes: (i) a brief description of the phenotype(s) associated with each mutation, (ii) whether a mutant phenotype has been detected by in vivo or in vitro methods, and (iii) relevant literature citations. The databases are available via ftp and on the World Wide Web at the following URL: http: //www.fandm.edu/Departments/Biology/Databases/RNA.h tml