RESUMO
BACKGROUND: The delivery of DNA into human cells has been the basis of advances in the understanding of gene function and the development of genetic therapies. Numerous chemical and physical approaches have been used to deliver the DNA, but their efficacy has been variable and is highly dependent on the cell type to be transfected. RESULTS: Studies were undertaken to evaluate and compare the transfection efficacy of several chemical reagents to that of the electroporation/nucleofection system using both adherent cells (primary and transformed airway epithelial cells and primary fibroblasts as well as embryonic stem cells) and cells in suspension (primary hematopoietic stem/progenitor cells and lymphoblasts). With the exception of HEK 293 cell transfection, nucleofection proved to be less toxic and more efficient at effectively delivering DNA into the cells as determined by cell proliferation and GFP expression, respectively. Lipofectamine and nucleofection of HEK 293 were essentially equivalent in terms of toxicity and efficiency. Transient transfection efficiency in all the cell systems ranged from 40%-90%, with minimal toxicity and no apparent species specificity. Differences in efficiency and toxicity were cell type/system specific. CONCLUSIONS: In general, the Amaxa electroporation/nucleofection system appears superior to other chemical systems. However, there are cell-type and species specific differences that need to be evaluated empirically to optimize the conditions for transfection efficiency and cell survival.
Assuntos
DNA/química , Transfecção/métodos , Animais , Linhagem Celular , Sobrevivência Celular , Eletroporação , Células-Tronco Embrionárias/metabolismo , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Humanos , Lipídeos/química , Camundongos , Polietilenoimina/química , Coelhos , SuínosRESUMO
An ultimate goal of gene therapy is the development of a means to correct mutant genomic sequences in the cells that give rise to pathology. A number of oligonucleotide-based gene-targeting strategies have been developed to achieve this goal. One approach, small fragment homologous replacement (SFHR), has previously demonstrated disease-specific genotypic and phenotypic modification after introduction of small DNA fragments (SDFs) into somatic cells. To validate whether the gene responsible for sickle cell anemia (beta-globin) can be modified by SFHR, a series of studies were undertaken to introduce sickle globin sequences at the appropriate locus of human hematopoietic stem/progenitor cells (HSPCs). The characteristic A two head right arrow T transversion in codon 6 of the beta-globin gene was indicated by restriction fragment length polymorphic (RFLP) analysis of polymerase chain reaction (PCR) products generated by amplification of DNA and RNA. At the time of harvest, it was determined that the cells generally contained =1 fragment per cell. Control studies mixing genomic DNA from nontransfected cells with varying amounts of the targeting SDFs did not indicate any PCR amplification artifacts due to the presence of residual SDF during amplification. RNA was analyzed after DNase treatment, thus eliminating the potential for SDF contamination. Stable SFHRmediated conversion of normal (beta (A)) to sickle (beta (S)) globin was detected at frequencies up to 13% in cells harvested 30-45 days posttransfection. The minimum conversion efficiency ranged from 0.2 to 3%, assuming modification of at least one cell per experiment showing conversion. Conversion of sickle (beta (S)) to normal (beta (A)) globin was detected up to 10 days posttransfection in lymphoblastoid cells from a sickle cell patient. These studies suggest that SFHR may be effective for ex vivo gene therapy of sickle cells in a patient's HSPCs before autologous transplantation.
Assuntos
Marcação de Genes , Globinas/genética , Células-Tronco Hematopoéticas/metabolismo , Oligodesoxirribonucleotídeos/metabolismo , Células Cultivadas , Sangue Fetal/citologia , Genoma Humano , Humanos , Oligodesoxirribonucleotídeos/genéticaRESUMO
Oligo/polynucleotide-based gene targeting strategies provide new options for achieving sequence-specific modification of genomic DNA and have implications for the development of new therapies and transgenic animal models. One such gene modification strategy, small fragment homologous replacement (SFHR), was evaluated qualitatively and quantitatively in human lymphoblasts that contain a single base substitution in the hypoxanthine-guanine phosphoribosyl transferase (HPRT1) gene. Because HPRT1 mutant cells are readily discernable from those expressing the wild type (wt) gene through growth in selective media, it was possible to identify and isolate cells that have been corrected by SFHR. Transfection of HPRT1 mutant cells with polynucleotide small DNA fragments (SDFs) comprising wild type HPRT1 (wtHPRT1) sequences resulted in clones of cells that grew in hypoxanthine-aminopterin-thymidine (HAT) medium. Initial studies quantifying the efficiency of correction in 3 separate experiments indicate frequencies ranging from 0.1% to 2%. Sequence analysis of DNA and RNA showed correction of the HPRT1 mutation. Random integration was not indicated after transfection of the mutant cells with an SDF comprised of green fluorescent protein (GFP) sequences that are not found in human genomic DNA. Random integration was also not detected following Southern blot hybridization analysis of an individual corrected cell clone.