Comparative transfection of DNA into primary and transformed mammalian cells from different lineages.

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.

Sequence-specific correction of genomic hypoxanthine-guanine phosphoribosyl transferase mutations in lymphoblasts by small fragment homologous replacement.

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.

Cl transport in complemented CF bronchial epithelial cells correlates with CFTR mRNA expression levels.

Little is known about the relationship between CF transmembrane conductance regulator (CFTR) gene expression and the corresponding transport of Cl. The phenotypic characteristics of polarized DeltaF508 homozygote CF bronchial epithelial (CFBE41o-) cells were evaluated following transfection with episomal expression vector containing either full-length (6.2kb) wild type (wt) and (4.7kb) DeltaF508CFTR cDNA. Forskolin-stimulated Cl secretion in two clones expressing the full-length wild type CFTR was assessed; clone c7-6.2wt gave 13.4+/-2.5 microA/cm(2) and clone c10-6.2wt showed 41.3+/-25.3 microA/cm(2). Another clone (c4-4.7DeltaF) complemented with the DeltaF508 CFTR cDNA showed high and stable expression of vector-derived DeltaF508 CFTR mRNA and a small cAMP-stimulated Cl current (4.7+/-0.7 microA/cm(2)) indicating DeltaF508CFTR trafficking to the plasma membrane at physiological temperatures. Vector-driven CFTR mRNA levels were 5-fold (c7-6.2wt), 14-fold (c10-6.2wt), and 27-fold (c7-4.7DeltaF) higher than observed in normal bronchial epithelial cells (16HBE14o-) endogenously expressing wtCFTR. Assessment of CFTR mRNA levels and CFTR function showed that cAMP-stimulated CFTR Cl currents were 33%, 167% and 24%, respectively, of those in 16HBE14o- cells. The data suggest that transgene expression needs to be significantly higher than endogenously expressed CFTR to restore functional wtCFTR Cl transport to levels sufficient to reverse CF pathology.

Gel purification of genomic DNA removes contaminating small DNA fragments interfering with polymerase chain reaction analysis of small fragment homologous replacement.

Oligonucleotides can mediate sequence-specific gene modification that results in the correction and/or alteration of genomic DNA. There is evidence to suggest that the polymerase chain reaction (PCR)-based analytical methods usually used to analyze oligonucleotide-mediated modification can generate artifacts. To investigate the conditions under which a PCR artifact can be generated and eliminated when analyzing small fragment homologous replacement (SHFR)-mediated modification, cells homozygous for the DeltaF508 mutation (CFBE41o-) were mixed with small DNA fragments (SDFs) containing the wild-type CFTR (wt-CFTR) sequence. An artifact could be generated after wild-type allele-specific PCR (wtAS-PCR) if the genomic DNA was not gel purified. Without gel purification, the amount of SDF/cell required to generate the artifact was dependent to the AS primer pairs used. When the genomic DNA was gel purified, no artifact could be detected with any of the wtAS-PCR primers whether the SDF was mixed with the cells or transfected into the cells. Furthermore, treatment of cellular mRNA with DNase was sufficient to eliminate potential artifacts in the reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. Thus, it is critical to gel purify genomic DNA and DNase treat mRNA when analyzing SFHR-mediated modification by PCR.

Modification of the pig CFTR gene mediated by small fragment homologous replacement.

The generation of a pig model of cystic fibrosis (CF) is a multistep process. Initial steps in this process involved the design and cloning of a small DNA fragment (SDF) or large oligodeoxynucleotide (LODN) that contains the F508del mutation and a silent restriction fragment length polymorphism causing mutation. This SDF/LODN was transfected into wild-type (WT) pig fetal fibroblast with the intention of modifying the pig genomic DNA by small fragment homologous replacement (SFHR). The targeted deletion (F508del) was detected in a subpopulation of transfected cells by allele-specific polymerase chain reaction (AS-PCR).

Small fragment homologous replacement-mediated modification of genomic beta-globin sequences in human hematopoietic stem/progenitor cells.

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

Characterization of novel airway submucosal gland cell models for cystic fibrosis studies.

Cultured airway epithelial cells are widely used in cystic fibrosis (CF) research as in vitro models that mimic the in vivo manifestations of the disease and help to define a specific cellular phenotype. Recently, a number of in vitro studies have used an airway adenocarcinoma cell line, Calu-3 that expresses submucosal gland cell features and significant levels of the wild-type CFTR mRNA and protein. We further characterized previously described CF tracheobronchial gland cell lines, CFSMEo- and 6CFSMEo- and determined that these cell lines are compound heterozygotes for the F508del and Q2X mutations, produce vestigial amounts of CFTR mRNA, and do not express detectable CFTR protein. Electrophysiologically, both cell lines are characteristically CF in that they lack cAMP-induced Cl- currents. In this study the cell lines are evaluated in the context of their role as the CF correlate to the Calu-3 cells. Together these cell systems provide defined culture systems to study the biology and pathology of CF. These airway epithelial cell lines may also be a useful negative protein control for numerous studies involving gene therapy by cDNA complementation or gene targeting.

Design of artificial nucleobases for the recognition of the AT inversion by triple-helix forming oligonucleotides: a structure-stability relationship study and neighbour bases effect.

We report herein on the synthesis, the incorporation into triplex forming oligonucleotides (TFO) and the recognition properties of a series of synthetic nucleosides designed for the specific recognition of an inverted A x T base pair in a pyrimidine triple helix motif. These analogues were designed on the basis of the results obtained with our previously reported compounds S and B(t), in order to define a structure-stability relationship. We report also on the chemical nature effect of the bases flanking S in the case of S-containing TFOs, in order to get further informations about the recognition process within the A x TxS triplet. This study establishes guidelines for the conception of more potent analogues for the recognition of both A x T and G x C inverted base pairs.

Synthesis, incorporation into triplex-forming oligonucleotide, and binding properties of a novel 2'-deoxy-C-nucleoside featuring a 6-(thiazolyl-5)benzimidazole nucleobase.

[reaction: see text] 6-(Thiazolyl-5)benzimidazole (B(t)()) was designed as a novel nucleobase for the specific recognition of an inverted A.T base pair in a triple helix motif. It was successfully incorporated into an 18-mer triplex-forming oligonucleotide (TFO) using the 2'-deoxy-C-nucleoside phosphoramidite 16. The triple helix binding properties of the modified TFO were examined by means of thermal denaturation experiments targeting an oligopyrimidine.oligopurine 26-mer DNA duplex containing an A.T base pair inversion.

Binding properties of oligonucleotides containing a modified 2'-deoxyuridine with a thymine ended linker to pair with 2'-deoxyadenosine.

Oligonucleotides containing in the place of thymidine the nucleoside 2, a 2'-deoxyuridine harbouring at C-5 a thymine ended linker, were found to undergo base pairing with the opposite 2'-deoxyadenosine. However, the corresponding duplexes are significantly destabilised as compared to the fully natural ones.